Capsid-modified, raav3 vector compositions and uses in gene therapy of human liver cancer

ABSTRACT

Disclosed are next-generation multi-mutated capsid protein-modified rAAV expression vectors, as well as infectious virions, compositions, and pharmaceutical formulations that include them. Also disclosed are methods of preparing and using these high transduction efficiency vector constructs in a variety of therapeutic applications including, inter alia, as delivery agents for the treatment or amelioration of one or more diseases or abnormal conditions in an affected mammal using in vivo and/or ex situ viral vector-based gene therapy protocols. Also disclosed are large-scale production methods for the multi-mutated, capsid-modified rAAV expression vectors, viral particles, and infectious virions, as well as use of the disclosed compositions in the manufacture of medicaments for use in a variety of in vitro and/or in vivo therapeutic methodologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present international patent application claims priority to U.S.patent application Ser. No. 13/899,481, filed May 21, 2013 (pending;Atty. Dkt. No. 36689.331), which was a continuation-in-part of U.S.patent application Ser. No. 12/595,196, filed Dec. 31, 2009 (now U.S.Pat. No. 8,445,267; Atty. Docket No. 36689.305), which was the U.S.national-stage filing of PCT Intl. Patent Appl. No. PCT/US2008/059647filed Apr. 8, 2008 (nationalized; Atty. Docket No. 36689.272), whichclaimed priority to U.S. Provisional Patent Appl. No. 60/910,798, filedApr. 9, 2007 (expired; Atty. Docket No. 36689.266). The content of eachof the aforementioned applications is hereby incorporated in itsentirety by express reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.R01-HL-097088 and R21-EB-015684 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularbiology and virology, and in particular, to the development of genedelivery vehicles. Also disclosed are improved rAAV vector compositionsuseful in delivering a variety of nucleic acid segments, including thoseencoding therapeutic proteins polypeptides, peptides, antisenseoligonucleotides, and ribozyme constructs to selected host cells for usein various diagnostic and/or therapeutic regimens. Methods are alsoprovided for preparing and using these modified rAAV-based vectorconstructs in a variety of viral-based gene therapies, and inparticular, for the diagnosis, prevention, treatment and/or ameliorationof symptoms of human diseases, disorders, dysfunctions, trauma, orinjury. The invention also provides mutated rAAV-based viral vectordelivery systems with increased transduction efficiency and/or improvedviral infectivity of selected mammalian host cells. In particular, theinvention provides improved rAAV vectors and virions having particleshaving amino acid substitutions in one or more surface-exposed residuesof a viral capsid protein.

2. Description of Related Art

Major advances in the field of gene therapy have been achieved by usingviruses to deliver therapeutic genetic material. The adeno-associatedvirus (AAV) has attracted considerable attention as a highly effectiveviral vector for gene therapy due to its low immunogenicity and abilityto effectively transduce non-dividing cells. AAV has been shown toinfect a variety of cell and tissue types, and significant progress hasbeen made over the last decade to adapt this viral system for use inhuman gene therapy.

In its normal “wild type” form, recombinant AAV (rAAV) DNA is packagedinto the viral capsid as a single stranded molecule about 4600nucleotides (nt) in length. Following infection of the cell by thevirus, the molecular machinery of the cell converts the single DNAstrand into a double-stranded form. Only the double-stranded DNA form isuseful to the polypeptides of the cell that transcribe the containedgene or genes into RNA.

AAV has many properties that favor its use as a gene deliveryvehicle: 1) the wild type virus is not associated with any pathologichuman condition; 2) the recombinant form does not contain native viralcoding sequences; and 3) persistent transgenic expression has beenobserved in many applications.

The transduction efficiency of recombinant adeno-associated virus 2(AAV) vectors varies greatly in different cells and tissues in vitro andin vivo, which has limited the usefulness of many of them in potentialgene therapy regimens. Systematic studies have been performed toelucidate the fundamental steps in the life cycle of AAV. For example,it has been documented that a cellular protein, FKBP52, phosphorylatedat tyrosine residues by epidermal growth factor receptor proteintyrosine kinase (EGFR-PTK), inhibits AAV second-strand DNA synthesis andconsequently, transgene expression in vitro as well as in vivo. It hasalso been demonstrated that EGFR-PTK signaling modulates theubiquitin/proteasome pathway-mediated intracellular trafficking as wellas FKBP52-mediated second-strand DNA synthesis of AAV vectors. In thosestudies, inhibition of EGFR-PTK signaling led to decreasedubiquitination of AAV capsid proteins, which in turn, facilitatednuclear transport by limiting proteasome-mediated degradation of AAVvectors, implicating EGFR-PTK-mediated phosphorylation of tyrosineresidues on AAV capsids. What is lacking in the prior art are improvedrAAV viral vectors that have enhanced transduction efficiency forinfecting selected mammalian cells, and for targeted gene delivery tohuman cells in particular.

BRIEF SUMMARY OF THE INVENTION

The present invention overcomes limitations and deficiencies inherent inthe prior art by providing novel improved rAAV-based genetic constructsthat encode one or more therapeutic agents useful in the preparation ofmedicaments for the prevention, treatment, and/or amelioration of one ormore diseases, disorders or dysfunctions resulting from a deficiency inone or more of such polypeptides. In particular, the invention providesVP3 capsid-protein-modified rAAV-based genetic constructs encoding oneor more selected molecules, such as, for example, one or more diagnosticor therapeutic agents (including, e.g., proteins, polypeptides,peptides, antibodies, antigen binding fragments, siRNAs, RNAis,antisense oligo- and poly-nucleotides, ribozymes, and variants and/oractive fragments thereof), for use in the diagnosis, prevention,treatment, and/or amelioration of symptoms of a variety of mammaliandiseases, disorders, dysfunctions, trauma, injury, and such like.

The present invention provides mutated AAV VP3 capsid proteins thatinclude modification of one or more surface-exposed amino acid resides(including, e.g., without limitation, lysine, serine, threonine, and/ortyrosine residues) as compared to wildtype. Also provided are infectiousrAAV virions that comprise the mutated AAV capsid proteins of thepresent invention, as well as nucleic acid molecules and rAAV vectorsencoding the mutant AAV capsid proteins of the present invention, andnucleic acids encoding one or more selected diagnostic and/ortherapeutic agents for delivery to a selected population of mammaliancells.

Advantageously, the novel rAAV vectors, express constructs, andinfectious virions and viral particles comprising them as disclosedherein preferably have an improved efficiency in transducing one or moreof a variety of cells, tissues and organs of interest, when compared towild-type, unmodified, expression constructs, and to the correspondingrAAV vectors and virions comprising them.

The improved rAAV vectors provided herein transduce one or more selectedhost cells at higher-efficiencies (and often much higher efficiencies)than conventional, wild type (i.e., “unmodified”) rAAV vectors. Byperforming extensive analysis and detailed experiments involving thesite-directed mutagenesis of various individual and/or combinations oftwo, three, four, five, or even six or more surface-exposed amino acidresidues on various AAV capsid proteins from a variety of AAV serotypes,the inventors have developed a large collection of single- ormulti-mutated rAAV vectors that possess improved transductionefficiencies. The inventors have demonstrated in a number of differentAAV serotypes that the substitution of one or more virionsurface-presenting amino acid residues results in improved viralvectors, which are capable of higher-efficiency transduction than thatof the corresponding, non-substituted vectors from which the mutantswere prepared.

The development of these new capsid-mutant rAAV viral vectorsdramatically reduces the number of viral particles needed forconventional gene therapy regimens. In addition to having improvedtransduction efficiencies for various mammalian cells, thesurface-exposed amino acid-modified rAAV vectors described herein aremore stable, less immunogenic, and can be produced at much lower costthan the traditional viral vectors currently employed in mammalian genetherapy regimens.

In a particular embodiment the invention provides a modified rAAV VP3capsid protein, that includes: (a) a non-tyrosine amino acid residue atone or more positions corresponding to Y252, Y272, Y444, Y701, Y705, andY731 of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3;(b) a non-serine amino acid residue at each of one or more positionscorresponding to S459 or 5663, of the wild-type AAV3 capsid protein asset forth in SEQ ID NO:3; (c) a non-threonine amino acid residue at eachof one or more positions corresponding to T251 or T492 of the wild-typeAAV3 capsid protein as set forth in SEQ ID NO:3; (d) a non-lysine aminoacid residue at each of one or more positions corresponding to K528,K533, or K545 of the wild-type AAV3 capsid protein as set forth in SEQID NO:3; (e) (i) a non-tyrosine amino acid residue at position Y701, orY705; and (ii) a non-tyrosine amino acid residue at position Y705 orY731, or a non-serine amino acid residue at position S663 of thewild-type AAV3 capsid protein as set forth in SEQ ID NO:3; (f) acombination of three or more amino acid substitutions listed in (a),(b), (c), and (d); each with a non-native amino acid; (g) a combinationof four or more amino acid substitutions listed in (a), (b), (c), and(d); each with a non-native amino acid; or (h) a combination of five ormore amino acid substitutions listed in (a), (b), (c), and (d); eachwith a non-native amino acid; or alternatively, wherein each of theamino acid substitutions is at an equivalent amino acid positioncorresponding thereto in any one of the other wild-type vector serotypesselected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV7,AAV8, AAV9, and AAV10, as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:10, respectively.

Exemplary multi-mutated proteins of the present invention include, butare not limited to, combinations of non-native amino acid substitutionsat each of three or more distinct surface-exposed amino acid residues onthe AAV3 capsid. These multi-mutant vectors include, without limitation:

(a) Y701F, Y705F, and Y731F; (b) Y705F, Y731F, and S663V; (c) Y705F,Y731F, and T492V; (d) Y705F, Y731F, K533R; (e) S663V, T492V, and K533R;(f) Y705F, Y731F, S663V, and T492V; and

(g) Y705F, Y731F, S663V, T492V, and K533R substitutions at the denotedamino acid residues of the wild-type AAV3 capsid protein as set forth inSEQ ID NO:3, or at the equivalent surface-exposed amino acid residues inany one of the corresponding wild-type AAV1, AAV2, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, or AAV10 capsid proteins, as set forth in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, or SEQ ID NO:10, respectively, or in any combinationthereof.

In the practice of the invention, the substituted non-native amino acidsmay include a substitution of one or more amino acids not normallypresent at a particular residue in the corresponding wild-type protein,and preferably include one or more non-native amino acid substitutionsselected from the group consisting of phenylalanine (F), valine (V),histidine (H), isoleucine (I), alanine (A), leucine (L) aspartic acid(D), asparagine (N). glutamic acid (E), arginine (R), serine (S), andisoleucine (I).

The invention also provides isolated and purified polynucleotides thatencode one or more of the disclosed capsid protein-mutated variants asdescribed herein, as well as recombinant adeno-associated viral (rAAV)vectors that comprise one or more such polynucleotides. Preferably, thevector constructs of the present invention further include at least onenucleic acid segment that encodes a diagnostic or therapeutic moleculeoperably linked to a promoter capable of expressing the nucleic acidsegment in a suitable host cell comprising the vector. In the practiceof the invention, the transduction efficiency of a virion comprising themodified AAV VP3 capsid protein will be higher than that of thecorresponding, unmodified, wild-type protein, and as such, willpreferably possess a transduction efficiency in a mammalian cell that isat least 2-fold, at least about 4-fold, at least about 6-fold, at leastabout 8-fold, at least about 10-fold, or at least about 12-fold orhigher in a selected mammalian host cell than that of a virion thatcomprises a corresponding, unmodified, capsid protein. In certainembodiments, the transduction efficiency of the rAAV vectors providedherein will be at least about 15-fold higher, at least about 20-foldhigher, at least about 25-fold higher, at least about 30-fold higher, orat least about 40, 45, or 50-fold or more greater than that of a virionthat comprises a corresponding, unmodified, capsid protein. Moreover,the infectious virions of the present invention that include one or moremodified AAV VP3 capsid proteins are preferably less susceptible toubiquitination when introduced into a mammalian cell than that of avirion that comprises a corresponding, unmodified, capsid protein.

The present invention also concerns rAAV vectors, wherein the nucleicacid segment further comprises a promoter, an enhancer, apost-transcriptional regulatory sequence, a polyadenylation signal, orany combination thereof, operably linked to the nucleic acid segmentthat encodes the selected polynucleotide of interest.

Preferably, the promoter is a heterologous promoter, a tissue-specificpromoter, a cell-specific promoter, a constitutive promoter, aninducible promoter, or any combination thereof.

In certain embodiments, the nucleic acid segments cloned into the novelrAAV expression vectors described herein will express or encode one ormore polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs,RNAis, antisense oligonucleotides, antisense polynucleotides,antibodies, antigen binding fragments, or any combination thereof.

As noted herein, the therapeutic agents useful in the invention mayinclude one or more agonists, antagonists, anti-apoptosis factors,inhibitors, receptors, cytokines, cytotoxins, erythropoietic agents,glycoproteins, growth factors, growth factor receptors, hormones,hormone receptors, interferons, interleukins, interleukin receptors,nerve growth factors, neuroactive peptides, neuroactive peptidereceptors, proteases, protease inhibitors, protein decarboxylases,protein kinases, protein kinase inhibitors, enzymes, receptor bindingproteins, transport proteins or one or more inhibitors thereof,serotonin receptors, or one or more uptake inhibitors thereof, serpins,serpin receptors, tumor suppressors, diagnostic molecules,chemotherapeutic agents, cytotoxins, or any combination thereof.

While the inventors particularly contemplate the use of the rAAV3vectors denoted in FIG. 41 in methods for the gene therapy of one ormore mammalian liver cancers, capsid-mutated vectors may be prepared andpackaged within virions of any known AAV serotype, including, forexamples, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 4(AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7(AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10(AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12).

The invention further provides populations and pluralities of suchcapsid-mutated rAAV vectors, as well as virions, infectious viralparticles, and mammalian host cells that include one or more nucleicacid segments encoding them.

Preferably, the mammalian host cells will be human host cells,including, for example blood cells, stem cells, hematopoietic cells,CD34⁺ cells, liver cells, cancer cells, vascular cells, pancreaticcells, neural cells, ocular or retinal cells, epithelial or endothelialcells, dendritic cells, fibroblasts, or any other cell of mammalianorigin, including, without limitation, hepatic (i.e., liver) cells, lungcells, cardiac cells, pancreatic cells, intestinal cells, diaphragmaticcells, renal (i.e., kidney) cells, neural cells, blood cells, bonemarrow cells, or any one or more selected tissues of a mammal for whichviral-based gene therapy is contemplated.

The invention further provides composition and formulations that includeone or more of the proteins nucleic acid segments viral vectors, hostcells, or viral particles of the present invention together with one ormore pharmaceutically-acceptable buffers, diluents, or excipients. Suchcompositions may be included in one or more diagnostic or therapeutickits, for diagnosing, preventing, treating or ameliorating one or moresymptoms of a mammalian disease, injury, disorder, trauma ordysfunction.

The invention further includes a method for providing a mammal in needthereof with a diagnostically- or therapeutically-effective amount of aselected biological molecule, the method comprising providing to a cell,tissue or organ of a mammal in need thereof, an amount of an rAAVvector; and for a time effective to provide the mammal with adiagnostically- or a therapeutically-effective amount of the selectedbiological molecule.

The invention further provides a method for diagnosing, preventing,treating, or ameliorating at least one or more symptoms of a disease, adisorder, a dysfunction, an injury, an abnormal condition, or trauma ina mammal. In an overall and general sense, the method includes at leastthe step of administering to a mammal in need thereof one or more of thedisclosed rAAV vectors, in an amount and for a time sufficient todiagnose, prevent, treat or ameliorate the one or more symptoms of thedisease, disorder, dysfunction, injury, abnormal condition, or trauma inthe mammal. In the case of rAAV3-based vectors, such abnormal conditionspreferably include one or more diseases or dysfunctions of the mammalianliver, including, for example, HCC; in the case of rAAV8-based vectors,such abnormal conditions preferably include one or more diseases ordysfunctions of the mammalian eye; or, in the case of rAAV6 vectors, oneor more diseases of stem cells, blood cells, hematopoietic cells, orCD35⁺ cells, including for example, sickle cell disease, β-thalassemia,and such like.

The invention also provides a method of transducing a population ofmammalian cells. In an overall and general sense, the method includes atleast the step of introducing into one or more cells of the population,a composition that comprises an effective amount of one or more of therAAV vectors disclosed herein.

In a further embodiment, the invention also provides isolated nucleicacid segments that encode one or more of the VP3 mutant capsid proteinsas described herein, and provides recombinant vectors, virus particles,infectious virions, and isolated host cells that comprise one or more ofthe improved vector sequences described and tested herein.

Additionally, the present invention provides compositions, as well astherapeutic and/or diagnostic kits that include one or more of thedisclosed vectors or AAv compositions, formulated with one or moreadditional ingredients, or prepared with one or more instructions fortheir use.

The invention also demonstrates methods for making, as well as methodsof using the disclosed improved rAAV capsid-mutated vectors in a varietyof ways, including, for example, ex situ, in vitro and in vivoapplications, methodologies, diagnostic procedures, and/or gene therapyregimens. Because many of the improved vectors described herein are alsoresistant to proteasomal degradation, they possess significantlyincreased transduction efficiencies in vivo making them particularlywell suited for viral vector-based human gene therapy regimens, and inparticular, for delivering one or more genetic constructs to selectedmammalian cells in vivo and/or in vitro.

In one aspect, the invention provides compositions comprisingrecombinant adeno-associated viral (AAV) vectors, virions, viralparticles, and pharmaceutical formulations thereof, useful in methodsfor delivering genetic material encoding one or more beneficial ortherapeutic product(s) to mammalian cells and tissues. In particular,the compositions and methods of the invention provide a significantadvancement in the art through their use in the treatment, prevention,and/or amelioration of symptoms of one or more mammalian diseases. It iscontemplated that human gene therapy will particularly benefit from thepresent teachings by providing new and improved viral vector constructsfor use in the treatment of a number of diverse diseases, disorders, anddysfunctions.

In another aspect, the invention concerns modified rAAV vector thatencode one or more mammalian therapeutic agents for the prevention,treatment, and/or amelioration of one or more disorders in the mammalinto which the vector construct is delivered.

In particular, the invention provides rAAV-based expression constructsthat encode one or more mammalian therapeutic agent(s) (including, butnot limited to, for example, protein(s), polypeptide(s), peptide(s),enzyme(s), antibodies, antigen binding fragments, as well as variants,and/or active fragments thereof, for use in the treatment, prophylaxis,and/or amelioration of one or more symptoms of a mammalian disease,dysfunction, injury, and/or disorder.

In one embodiment, the invention provides an rAAV vector that comprisesat least a first capsid protein comprising at least a first amino acidsubstitution to a non-native amino acid at one or more surface exposedamino acid residues in an rAAV capsid protein, and wherein the vectorfurther additionally includes at least a first nucleic acid segment thatencodes at least a first diagnostic or therapeutic agent operably linkedto a promoter capable of expressing the segment in a host cell thatcontains the expression vector construct.

The surface-exposed amino acid-modified rAAV vectors of the presentinvention may optionally further include one or more enhancer sequencesthat are each operably linked to the nucleic acid segment. Exemplaryenhancer sequences include, but are not limited to, one or more selectedfrom the group consisting of a CMV enhancer, a synthetic enhancer, aliver-specific enhancer, an vascular-specific enhancer, a brain-specificenhancer, a neural cell-specific enhancer, a lung-specific enhancer, amuscle-specific enhancer, a kidney-specific enhancer, apancreas-specific enhancer, and an islet cell-specific enhancer.

Exemplary promoters useful in the practice of the invention include,without limitation, one or more heterologous, tissue-specific,constitutive or inducible promoters, including, for example, but notlimited to, a promoter selected from the group consisting of a CMVpromoter, a β-actin promoter, an insulin promoter, an enolase promoter,a BDNF promoter, an NGF promoter, an EGF promoter, a growth factorpromoter, an axon-specific promoter, a dendrite-specific promoter, abrain-specific promoter, a hippocampal-specific promoter, akidney-specific promoter, an elafin promoter, a cytokine promoter, aninterferon promoter, a growth factor promoter, an α₁-antitrypsinpromoter, a brain cell-specific promoter, a neural cell-specificpromoter, a central nervous system cell-specific promoter, a peripheralnervous system cell-specific promoter, an interleukin promoter, a serpinpromoter, a hybrid CMV promoter, a hybrid β-actin promoter, an EF1promoter, a U1a promoter, a U1b promoter, a Tet-inducible promoter, aVP16-LexA promoter, or any combination thereof. In exemplaryembodiments, the promoter may include a mammalian or avian β-actinpromoter.

The first nucleic acid segment may also further include one or morepost-transcriptional regulatory sequences or one or more polyadenylationsignals, including, for example, but not limited to, a woodchuckhepatitis virus post-transcription regulatory element, a polyadenylationsignal sequence, or any combination thereof.

Exemplary diagnostic or therapeutic agents deliverable to host cells bythe present vector constructs include, but are not limited to, an agentselected from the group consisting of a polypeptide, a peptide, anantibody, an antigen binding fragment, a ribozyme, a peptide nucleicacid, a siRNA, an RNAi, an antisense oligonucleotide, an antisensepolynucleotide, and any combination thereof.

In exemplary embodiments, the improved rAAV vectors of the inventionwill preferably encode at least one diagnostic or therapeutic protein orpolypeptide selected from the group consisting of a molecular marker, anadrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, acytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, aglutamic acid decarboxylase, a glycoprotein, a growth factor, a growthfactor receptor, a hormone, a hormone receptor, an interferon, aninterleukin, an interleukin receptor, a kinase, a kinase inhibitor, anerve growth factor, a netrin, a neuroactive peptide, a neuroactivepeptide receptor, a neurogenic factor, a neurogenic factor receptor, aneuropilin, a neurotrophic factor, a neurotrophin, a neurotrophinreceptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, aprotease inhibitor, a protein decarboxylase, a protein kinase, a proteinkinase inhibitor, a proteolytic protein, a proteolytic proteininhibitor, a semaphorin, a semaphorin receptor, a serotonin transportprotein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, aserpin receptor, a tumor suppressor, and any combination thereof.

In certain applications, the capsid-modified rAAV vectors of the presentinvention may include one or more nucleic acid segments that encode apolypeptide selected from the group consisting of BDNF, CNTF, CSF, EGF,FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF,TGF, TGF-B2, TNF, VEGF, prolactin, somatotropin, XIAP1, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(I87A), viralIL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and anycombination thereof.

In another embodiment, the invention concerns genetically-modifiedimproved transduction-efficiency rAAV vectors that include at least afirst nucleic acid segment that encodes one or more therapeutic agentsthat alter, inhibit, reduce, prevent, eliminate, or impair the activityof one or more endogenous biological processes in the cell. Inparticular embodiments, such therapeutic agents may be those thatselectively inhibit or reduce the effects of one or more metabolicprocesses, dysfunctions, disorders, or diseases. In certain embodiments,the defect may be caused by injury or trauma to the mammal for whichtreatment is desired. In other embodiments, the defect may be caused theover-expression of an endogenous biological compound, while in otherembodiments still; the defect may be caused by the under-expression oreven lack of one or more endogenous biological compounds.

When the use of such vectors is contemplated for introduction of one ormore exogenous proteins, polypeptides, peptides, ribozymes, siRNAs,and/or antisense oligonucleotides, to a particular cell transfected withthe vector, one may employ the modified AAV vectors disclosed herein byincorporating into the vector at least a first exogenous polynucleotideoperably positioned downstream and under the control of at least a firstheterologous promoter that expresses the polynucleotide in a cellcomprising the vector to produce the encoded therapeutic agent,including for example, peptides, proteins, polypeptides, antibodies,ribozymes, siRNAs, and antisense oligo- or polynucleotides.

The genetically-modified rAAV vectors and expression systems of thepresent invention may also further optionally include a second distinctnucleic acid segment that comprises, consists essentially of, orconsists of, one or more enhancers, one or more regulatory elements, oneor more transcriptional elements, or any combination thereof, thatalter, improve, regulate, and/or affect the transcription of thenucleotide sequence of interest expressed by the modified rAAV vectors.

For example, the rAAV vectors of the present invention may furtherinclude a second nucleic acid segment that comprises, consistsessentially of, or consists of, a CMV enhancer, a synthetic enhancer, acell-specific enhancer, a tissue-specific enhancer, or any combinationthereof. The second nucleic acid segment may also further comprise,consist essentially of, or consist of, one or more intron sequences, oneor more post-transcriptional regulatory elements, or any combinationthereof.

The improved vectors and expression systems of the present invention mayalso optionally further include a polynucleotide that comprises,consists essentially of, or consists of, one or more polylinkers,restriction sites, and/or multiple cloning region(s) to facilitateinsertion (cloning) of one or more selected genetic elements, genes ofinterest, or therapeutic or diagnostic constructs into the rAAV vectorat a selected site within the vector.

In further aspects of the present invention, the exogenouspolynucleotide(s) that may be delivered into suitable host cells by theimproved, capsid-modified, rAAV vectors disclosed herein are preferablyof mammalian origin, with polynucleotides encoding one or morepolypeptides or peptides of human, non-human primate, porcine, bovine,ovine, feline, canine, equine, epine, caprine, or lupine origin beingparticularly preferred.

The exogenous polynucleotide(s) that may be delivered into host cells bythe disclosed capsid-modified viral vectors may, in certain embodiments,encode one or more proteins, one or more polypeptides, one or morepeptides, one or more enzymes, or one or more antibodies (orantigen-binding fragments thereof), or alternatively, may express one ormore siRNAs, ribozymes, antisense oligonucleotides, PNA molecules, orany combination thereof. When combinational gene therapies are desired,two or more different molecules may be produced from a single rAAVexpression system, or alternatively, a selected host cell may betransfected with two or more unique rAAV expression systems, each ofwhich may comprise one or more distinct polynucleotides that encode atherapeutic agent.

In other embodiments, the invention also provides capsid-modified rAAVvectors that are comprised within an infectious adeno-associated viralparticle or a virion, as well as pluralities of such virions orinfectious particles. Such vectors and virions may be comprised withinone or more diluents, buffers, physiological solutions or pharmaceuticalvehicles, or formulated for administration to a mammal in one or morediagnostic, therapeutic, and/or prophylactic regimens. The vectors,virus particles, virions, and pluralities thereof of the presentinvention may also be provided in excipient formulations that areacceptable for veterinary administration to selected livestock, exotics,domesticated animals, and companion animals (including pets and suchlike), as well as to non-human primates, zoological or otherwise captivespecimens, and such like.

The invention also concerns host cells that comprise at least one of thedisclosed capsid protein-modified rAAV expression vectors, or one ormore virus particles or virions that comprise such an expression vector.Such host cells are particularly mammalian host cells, with human hostcells being particularly highly preferred, and may be either isolated,in cell or tissue culture. In the case of genetically modified animalmodels, the transformed host cells may even be comprised within the bodyof a non-human animal itself.

In certain embodiments, the creation of recombinant non-human hostcells, and/or isolated recombinant human host cells that comprise one ormore of the disclosed rAAV vectors is also contemplated to be useful fora variety of diagnostic, and laboratory protocols, including, forexample, means for the production of large-scale quantities of the rAAVvectors described herein. Such virus production methods are particularlycontemplated to be an improvement over existing methodologies includingin particular, those that require very high titers of the viral stocksin order to be useful as a gene therapy tool. The inventors contemplatethat one very significant advantage of the present methods will be theability to utilize lower titers of viral particles in mammaliantransduction protocols, yet still retain transfection rates at asuitable level.

Compositions comprising one or more of the disclosed capsid-modified,improved transduction-efficiency rAAV vectors, expression systems,infectious AAV particles, or host cells also form part of the presentinvention, and particularly those compositions that further comprise atleast a first pharmaceutically-acceptable excipient for use in therapy,and for use in the manufacture of medicaments for the treatment of oneor more mammalian diseases, disorders, dysfunctions, or trauma. Suchpharmaceutical compositions may optionally further comprise one or morediluents, buffers, liposomes, a lipid, a lipid complex; or thetyrosine-modified rAAV vectors may be comprised within a microsphere ora nanoparticle.

Pharmaceutical formulations suitable for intramuscular, intravenous, ordirect injection into an organ or tissue or a plurality of cells ortissues of a human or other mammal are particularly preferred, however,the compositions disclosed herein may also find utility inadministration to discreet areas of the mammalian body, including forexample, formulations that are suitable for direct injection into one ormore organs, tissues, or cell types in the body. Such injection sitesinclude, but are not limited to, the brain, a joint or joint capsule, asynovium or subsynovium tissue, tendons, ligaments, cartilages, bone,peri-articular muscle or an articular space of a mammalian joint, aswell as direct administration to an organ such as the heart, liver,lung, pancreas, intestine, brain, bladder, kidney, or other site withinthe patient's body, including, for example, introduction of the viralvectors via intraabdominal, intrathorascic, intravascular, orintracerebroventricular delivery.

Other aspects of the invention concern recombinant adeno-associatedvirus virion particles, compositions, and host cells that comprise,consist essentially of, or consist of, one or more of thecapsid-modified, improved transduction efficiency, rAAV vectorsdisclosed herein, such as for example pharmaceutical formulations of thevectors intended for administration to a mammal through suitable means,such as, by intramuscular, intravenous, intra-articular, or directinjection to one or more cells, tissues, or organs of a selected mammal.Typically, such compositions may be formulated withpharmaceutically-acceptable excipients as described hereinbelow, and maycomprise one or more liposomes, lipids, lipid complexes, microspheres ornanoparticle formulations to facilitate administration to the selectedorgans, tissues, and cells for which therapy is desired.

Kits comprising one or more of the disclosed capsid-modified rAAVvectors (as well as one or more virions, viral particles, transformedhost cells or pharmaceutical compositions comprising such vectors); andinstructions for using such kits in one or more therapeutic, diagnostic,and/or prophylactic clinical embodiments are also provided by thepresent invention. Such kits may further comprise one or more reagents,restriction enzymes, peptides, therapeutics, pharmaceutical compounds,or means for delivery of the composition(s) to host cells, or to ananimal (e.g., syringes, injectables, and the like). Exemplary kitsinclude those for treating, preventing, or ameliorating the symptoms ofa disease, deficiency, dysfunction, and/or injury, or may includecomponents for the large-scale production of the viral vectorsthemselves, such as for commercial sale, or for use by others, includinge.g., virologists, medical professionals, and the like.

Another important aspect of the present invention concerns methods ofuse of the disclosed rAAV vectors, virions, expression systems,compositions, and host cells described herein in the preparation ofmedicaments for diagnosing, preventing, treating or ameliorating atleast one or more symptoms of a disease, a dysfunction, a disorder, anabnormal condition, a deficiency, injury, or trauma in an animal, and inparticular, in a vertebrate mammal. Such methods generally involveadministration to a mammal in need thereof, one or more of the disclosedvectors, virions, viral particles, host cells, compositions, orpluralities thereof, in an amount and for a time sufficient to diagnose,prevent, treat, or lessen one or more symptoms of such a disease,dysfunction, disorder, abnormal condition, deficiency, injury, or traumain the affected animal. The methods may also encompass prophylactictreatment of animals suspected of having such conditions, oradministration of such compositions to those animals at risk fordeveloping such conditions either following diagnosis, or prior to theonset of symptoms.

As described above, the exogenous polynucleotide will preferably encodeone or more proteins, polypeptides, peptides, ribozymes, or antisenseoligonucleotides, or a combination of these. In fact, the exogenouspolynucleotide may encode two or more such molecules, or a plurality ofsuch molecules as may be desired. When combinational gene therapies aredesired, two or more different molecules may be produced from a singlerAAV expression system, or alternatively, a selected host cell may betransfected with two or more unique rAAV expression systems, each ofwhich will provide unique heterologous polynucleotides encoding at leasttwo different such molecules.

Compositions comprising one or more of the disclosed rAAV vectors,expression systems, infectious AAV particles, host cells also form partof the present invention, and particularly those compositions thatfurther comprise at least a first pharmaceutically-acceptable excipientfor use in the manufacture of medicaments and methods involvingtherapeutic administration of such rAAV vectors. Such pharmaceuticalcompositions may optionally further comprise liposomes, a lipid, a lipidcomplex; or the rAAV vectors may be comprised within a microsphere or ananoparticle. Pharmaceutical formulations suitable for intramuscular,intravenous, or direct injection into an organ or tissue of a human areparticularly preferred.

Another important aspect of the present invention concerns methods ofuse of the disclosed vectors, virions, expression systems, compositions,and host cells described herein in the preparation of medicaments fortreating or ameliorating the symptoms of various polypeptidedeficiencies in a mammal. Such methods generally involve administrationto a mammal, or human in need thereof, one or more of the disclosedvectors, virions, host cells, or compositions, in an amount and for atime sufficient to treat or ameliorate the symptoms of such a deficiencyin the affected mammal. The methods may also encompass prophylactictreatment of animals suspected of having such conditions, oradministration of such compositions to those animals at risk fordeveloping such conditions either following diagnosis, or prior to theonset of symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

For promoting an understanding of the principles of the invention,reference will now be made to the embodiments, or examples, illustratedin the drawings and specific language will be used to describe the same.It will, nevertheless be understood that no limitation of the scope ofthe invention is thereby intended. Any alterations and furthermodifications in the described embodiments, and any further applicationsof the principles of the invention as described herein are contemplatedas would normally occur to one of ordinary skill in the art to which theinvention relates.

The following drawings form part of the present specification and areincluded to demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to the followingdescription taken in conjunction with the accompanying drawings, inwhich like reference numerals identify like elements, and in which:

FIG. 1A, FIG. 1B, and FIG. 1C show the effect of NF-κB pathwayinhibitors and activator on AAV vector-mediated EGFP expression in HeLacells in vitro. Cells were pre-treated with various concentrations ofinhibitors and activators for 12 hrs and transduced with 2×10³ AAV-EGFPvgs per cell. FIG. 1A shows the transgene expression was detected byfluorescence microscopy 48 hrs post-infection. Representative images areshown. FIG. 1B shows the quantitative analyses of the data from FIG. 1A.Images from five visual fields were analyzed as described. *P<0.001.FIG. 1C is a Western blot analysis of HeLa cell extracts transduced withscAAV vectors and in the presence of NF-κB modulators. The samples wereanalyzed by using anti-p65 and anti-IκB antibodies [classical pathway],anti-p100/p52 antibody [non-canonical pathway] for detection NF-κBsignaling in response to AAV exposure. These results are representativeof two independent experiments;

FIG. 2A and FIG. 2B show AAV-EGFP vector-mediated transduction ofprimary human monocytes-derived dendritic cells in the presence of NF-κBmodulators. FIG. 2A shows the transgene expression was detected by flowcytometry 48 hrs post-transduction. FIG. 2B is a Western blot analysisfor components of classical and non-canonical pathway of NF-κBactivation in nuclear extracts from dendritic cells, mock-transduced ortransduced with 2,000 vgs/cell of scAAV vectors and in the presence ofNF-κB modulators;

FIG. 3A and FIG. 3B show AAV vector-induced innate immune and NF-κBresponse in mice in vivo. Gene expression profiling of innate immunemediators (FIG. 3A) or NF-κB activation (FIG. 3B) was performed asdescribed. The data for fold changes in gene expression at the 2-hrtime-point comparing AAV vectors with Bay11 (hatched or open bars) withAAV vectors without Bay11 (black or grey bars) are shown. The minimalthreshold fold-increase (horizontal black line) was 2.5 (FIG. 3A) or 3.0(FIG. 3B) by measuring the variability of duplicate ΔCT (compared toGAPDH, 2̂^(−ΔCT(variability)));

FIG. 4A and FIG. 4B illustrate transgene expression in murinehepatocytes 10 days post-injection of 1×10¹¹ vgs each of WT-scAAV-EGFPor TM-scAAV-EFGP vectors/animal via the tail-vein. FIG. 4A showsrepresentative images are shown. Original magnification: ×400. FIG. 4Bshows the quantitative analyses of the data from FIG. 4A. Images fromfive visual fields were analyzed quantitatively as described in thelegend to FIG. 1A;

FIG. 5 demonstrates that AAV genome contains putative binding sites forNF-κB-responsive transcription factors within the inverted terminalrepeats (ITRs). The putative NF-κB-responsive transcriptionfactor-binding sites in the AAV-ITRs were identified by in silicoanalysis using the web-based TRANSFAC database. The binding sites forp300, TFIIB, and SpII transcriptions factors are denoted by green, red,and blue underlined fonts, respectively. The boxed sequence representsthe 20-nucleotide, single-stranded D-sequence within the ITR;

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, and FIG. 6E show the effect of NF-κBactivators and inhibitors on transgene expression from an AAV2-EGFPvector in HeLa cells in vitro. Cells were either mock-treated orpretreated with various combinations of inhibitors and activators for 12hr. Washed cells were infected with 2×10³ vg/cell of scAAV2-EGFP (FIG.6A), ssAAV2-EGFP (FIG. 6B), or TM-scAAV2-EGFP (FIG. 6C). Transgeneexpression was detected by fluorescence microscopy 48-hrs'postinfection. Representative images are shown; Western blot analysis ofcytoplasmic (FIG. 6D) and nuclear (FIG. 6E) extracts from HeLa cellstransduced with scAAV vectors and in the presence of NF-κB modulators.The samples were analyzed by using anti-p100/p52 antibody for detectionof NF-κB signaling. Anti-GAPDH and lamin B antibodies were used asappropriate controls. These results are representative of twoindependent experiments;

FIG. 7 shows a Western blot analysis of liver homogenates from micefollowing mock-injections (n=2), or injections with scAAV vectors, withand without prior administration of Bay11 (n=3 each). The samples wereanalyzed by using anti-p52 antibody for detection NF-κB signaling inresponse to AAV exposure. Anti-β-actin antibody was used as a loadingcontrol;

FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, and FIG. 8F show foldchanges in gene expression of various cytokines/chemokines from totalmRNA collected from liver samples from animals injected with the WT-AAVor the TM-AAV vectors, following PBS- or Bay11-pre-treatment. FIG. 8A:IL-1α; FIG. 8B: IL-6; FIG. 8C: TNF-α; FIG. 8D: IL-12α, FIG. 8E: KC; andFIG. 8F: RANTES. Values are significant above 2.6 and below 0.38;calculated by determining the variability in the 96-well plates used tomeasure specific gene expression;

FIG. 9 demonstrates humoral response to AAV vectors in the absence orpresence of NF-kB inhibitor. Anti-AAV2 IgG2a levels were determined inperipheral blood from mice at day 10 following injections with scAAVvectors, with and without prior administration of Bay11 (n=4 each);

FIG. 10 illustrate electrophoretic mobility-shift assays carried outwith whole-cell extracts prepared from HeLa cells and ³²P-labeledsingle-stranded D[+]-sequence probe (lane 1), which interacted with ahost cell protein (lane 3, arrowhead). Single-stranded D[−]-sequence(lane 2) probe was used as an appropriate control, which also interactedwith a cellular protein, FKBP52 (lane 4, arrow). Binding assays werealso carried out using biotin-labeled ssD[+]-sequence probe followed byselection with streptavidin-beads, and fractionation bySDS-polyacrylamide gel electrophoresis. The relevant protein band wasvisualized by silver staining, excised from the gel, and subjected tomass spectrometry, and one of the unique peptides was found to sharehomology with the NF-κB-repressing factor (NRF);

FIG. 11A and FIG. 11B show the analysis of AAV3-mediated transgeneexpression in T47D and T47D+hHGFR cells. FIG. 11A shows equivalentnumbers of T47D and T47D+hHGFR cells were infected with variousindicated multiplicity-of-infection (MOI) of scAAV3-CBAp-EGFP vectorsunder identical conditions. Transgene expression was determined byfluorescence microscopy 72 hrs post-infection. FIG. 11B shows T47D+hHGFRcells were transduced with 2,000 vgs/cell of scAAV3 vectors in theabsence or the presence of 5 μg/mL of hHGF. Transgene expression wasdetermined by fluorescence microscopy 72-hrs' post-infection;

FIG. 12A, FIG. 12B and FIG. 12C show the effect of BMS-777607 onAAV3-mediated transgene expression. FIG. 12A shows T47D+hHGFR cells,either mock-treated or treated with various concentration of BMS-777607,that were infected with 2,000 vgs/cell of scAAV3-CBAp-EGFP vectors.Transgene expression was determined by fluorescence microscopy 72 hrs'post-infection. FIG. 12B illustrates T47D and T47D+hHGFR cells wereinfected with 10,000 vgs/cell of scAAV3-CBAp-EGFP vectors in the absenceor the presence of 1 μM of BMS-777607. FIG. 12C shows T47D andT47D+hHGFR cells were mock-treated or pretreated with BMS-777607 for twohrs. Whole-cell lysates were prepared and analyzed on Western blotsusing various indicated primary antibodies. β-actin was used as aloading control;

FIG. 13A and FIG. 13B show the effect of BMS-777607 on various AAVserotype-mediated transgene expression. In FIG. 13A, T47D+hHGFR cells,either mock-treated or treated with 1 μM of BMS-777607, were infectedwith 2,000 vgs/cell of either scAAV2-, scAAV3- or scAAV4-CBAp-EGFPvectors. In FIG. 13B, T47D+hHGFR cells, either mock-treated or treatedwith 1 μM of BMS-777607, were infected with 2,000 vgs/cell of eitherscAAV5-, scAAV7-, scAAV8- or scAAV9-CBAp-EGFP vectors. Transgeneexpression was determined by fluorescence microscopy 72 hrspost-infection;

FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14D show the comparative analysesof AAV3-mediated transduction efficiency in Huh7 and Hep293TT cells withor without treatment with MG132. In FIG. 14A, HeLa cells, eithermock-treated or treated with 5 μM of MG132, were infected withscAAV2-CBAp-EGFP vectors. In FIG. 14B, Huh7 and Hep293TT cells, eithermock-treated or treated with various concentration of MG132, wereinfected with scAAV3-WT-CBAp-EGFP vectors. In FIG. 14C, HeLa cells,either mock-treated or treated with 200 μM of Tyr23, were infected byscAAV2-CBAp-EGFP vectors. In FIG. 14D, Hep293TT cells, eithermock-treated or treated with Tyr23, were infected by scAAV3-CBAp-EGFPvectors. Transgene expression was determined 72 hrs' post-transduction;

FIG. 15A, FIG. 15B and FIG. 15C show the site-directed mutationalanalyses of surface-exposed tyrosine residues on AAV3 capsids. Huh7cells were transduced with WT or F501Y scAAV3-CBAp-EGFP vectors underidentical conditions, and transgene expression was determined 72 hrs'post-transduction. Transduction efficiency of WT (FIG. 15A) and variousY-F scAAV3-mediated transgene expression in Huh7 (FIG. 15B) and Hep293TT(FIG. 15C) cells are shown. Transgene expression was determined 72 hrspost-transduction;

FIG. 16A, FIG. 16B, and FIG. 16C illustrate the transduction efficiencyof WT and single, double, and triple tyrosine-mutant AAV3 vectors. InFIG. 16A, Huh7 cells were transduced with WT or various indicated Y-Fmutant scAAV3-CBAp-EGFP vectors under identical conditions. Transgeneexpression was determined 72-hrs' post-transduction. In FIG. 16B, Huh7cells were transduced with 5,000 vgs/cell of WT or Y→F mutated scAAV3vectors in the absence or the presence of 5 μg/mL of hHGF. FIG. 16Cshows transgene expression was determined by fluorescence microscopy 72hrs post-infection;

FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D show the transductionefficiency of AAV3 vectors in vivo following direct intra-tumorinjections. Transduction efficiency of WT-AAV3 vectors in Huh7- (FIG.17A) and Hep293TT- (FIG. 17B) derived tumors in NSG mice is shown. Thetransduction efficiency of WT- (FIG. 17C) and Y705+731F-AAV3 (FIG. 17D)vectors in Hep293TT-derived tumors are also shown in NSG mice. EGFPfluorescence (green) and DAPI staining (blue) of two representativetumor sections from each set of mice is shown;

FIG. 18A, FIG. 18B and FIG. 18C illustrate the transduction efficiencyof WT- and Y705+731F-AAV3 vectors in Hep293TT-derived tumors in NSG micefollowing tail-vein injections. EGFP fluorescence (green) and DAPIstaining (blue) of tumor in three representative tumor sections fromeach set of mice injected with PBS (FIG. 18A), WT-AAV3 (FIG. 18B), orY705+731F-AAV3 (FIG. 18C) vectors is shown;

FIG. 19A and FIG. 19B show the effect of various kinase inhibitors onssAAV and scAAV mediated EGFP expression in HEK293 cells. Cells werepretreated with inhibitors for 1 hr before infection then transducedwith 1×10³ vgs/cell. In FIG. 19A, transgene expression was detected byfluorescence microscopy 48 hrs post infection. In FIG. 19B, images fromthree visual fields were analyzed as described. *P<0.005, **P<0.001 vs.WT AAV2;

FIG. 20A and FIG. 20B show the analysis of EGFP expression aftertransduction of HEK293 cells with individual site-directed scAAV2 capsidmutants. Each of the 15 surface-exposed serines (S) in the AAV2 capsidwas substituted with valine (V), and then evaluated for its efficiencyto mediate transgene expression. FIG. 20A shows the EGFP expressionanalysis at 48 hrs post-infection at an MOI of 1×10³ vgs/cell. FIG. 20Bshows the quantitation of transduction efficiency of each of theserine-mutant AAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2;

FIG. 21 and FIG. 21B illustrate the structure of AAV2. In FIG. 21A, atrimer of the AAV2 VP3 shown in ribbon representation and viewed downthe icosahedral threefold axis (left) and rotated 90° (right) with VPmonomers colored in blue, purple and light blue showing the location ofserine residues 458, 492, and 662 in the yellow, green, and red spheres,respectively. The approximate positions of the icosahedral two-, three-,and five-fold axes are depicted by the filled oval, triangle, andpentagon, respectively. In FIG. 21B, the capsid surface of AAV2 shown inblue with serine residues 458, 492, and 662 highlighted in the samecolors as shown previously in similar figures. The S458 and S492residues are located adjacent to each other on the outer surface of theprotrusions facing the depression surrounding the two-fold axes. S662 islocated on the HI loop (colored white) (between the β-H and β-I strandsof the core eight-stranded beta-barrel) which lie on the floor of thedepression surrounding the icosahedral five-fold axes. The five-foldsymmetry related DE loops (between the β-D and β-E strands), which formthe channel at the icosahedral 5-fold axes, are shown in brown. Theapproximate positions of an icosahedral two-fold (2F), three-fold (3F),and five-fold (5F) axes are indicated by the open arrows;

FIG. 22A and FIG. 22B summarize the evaluation of the effect of serinesubstitution at position 662 in the scAAV2 capsid with different aminoacids in mediating transgene expression. The following eight serinemutants were generated with different amino acids: S662→Valine (V),S662→Alanine (A), S662→Asparagine (N), S662→Aspartic acid (D),S662→Histidine (H), S662→Isoleucine (I), S662→Leucine (L), andS662→Phenylalanine (F), and their transduction efficiency in 293 cellswas analyzed. FIG. 22A shows the EGFP expression analysis at 48 h afterinfection of 293 cells at an MOI of 1×10³ vgs/cell. FIG. 22B shows thequantitation of the transduction efficiency of each of the serine-mutantAAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2;

FIG. 23A and FIG. 23B show the analysis of correlation of transductionefficiency of scAAV2-S662V vectors with p38 MAPK activity in variouscell types. FIG. 23A illustrates the quantitation of the transductionefficiency of WT- and S662V-AAV2 vectors in HEK293, HeLa, NIH3T3, H2.35and moDCs. FIG. 23B is a Western blot analysis of lysates from differentcell lines for p-p38 MAPK expression levels. Total p38 MAPK and GAPDHlevels were measured and used as loading controls. *P<0.005, **P<0.001vs. WT AAV2;

FIG. 24A, FIG. 24B, and FIG. 24C illustrate scAAV vector-mediatedtransgene expression in monocyte-derived dendritic cells (moDCs). FIG.24A illustrates the effect of JNK and p38 MAPK inhibitors, andsite-directed substitution of the serine residue at position 662 on EGFPexpression. FIG. 24B summarizes the quantitation of data from FIG. 24Aat 48 hrs after infection and initiation of maturation. FIG. 24C is ananalysis of expression of co-stimulatory markers such as CD80, CD83,CD86 in moDCs infected with scAAV2-S662V vectors at an MOI of 5×10⁴vgs/cell. iDCs—immature dendritic cells, and mDCs—mature dendriticcells, stimulated with cytokines, generated as described herein, wereused as negative and positive controls, respectively. A representativeexample is shown. *P<0.005, **P<0.001 vs. WT AAV2;

FIG. 25 illustrates analysis of hTERT-specific cytotoxic T-lymphocytes(CTLs) killing activity on K562 cells. CTLs were generated aftertransduction of moDCs by scAAV2-S662V vectors encoding the truncatedhuman telomerase (hTERT). scAAV2-S662V-EGFP vector-traduced moDCs wereused to generate non-specific CTLs. Pre-stained with3,3-dioctadecyloxacarbocyanine (DiOC18(3)), a green fluorescent membranestain, 1×10⁵ target K562 cells were co-cultured overnight with differentratios of CTLs (80:1, 50:1, 20:1, 10:1, 5:1). Membrane-permeable nucleicacid counter-stain, propidium iodide, was added to label the cells withcompromised plasma membranes. Percentages of killed, doublestain-positive cells were analyzed by flow cytometry;

FIG. 26A and FIG. 26B show the analysis of EGFP expression aftertransduction of HEK293 cells with individual site-directed AAV2 capsidmutants. Each of the 17 surface-exposed threonine (T) residues in AAV2capsid was substituted with valine (V) and evaluated for its efficiencyto mediate transgene expression. In FIG. 26A, EGFP expression analysisat 48-hrs' post-infection is shown (MOI of 1×10³ vg/cell). FIG. 26Bshows the quantification of transduction efficiency of each of thethreonine-mutant scAAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2;

FIG. 27A and FIG. 27B show the analysis of EGFP expression in HEK293cells infected with multiple site-directed AAV2 capsid mutants. Severalmost efficient threonine mutations were combined on single AAV2 capsidto produce double- and triple-mutant and efficiency of each vector wasevaluated. FIG. 27A illustrates EGFP expression analysis at 48 hrs'post-infection at MOI of 1×10³ vg/cell. FIG. 27B shows thequantification of transduction efficiency of each of thethreonine-mutant AAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2;

FIG. 28A and FIG. 28B demonstrate the evaluation of EGFP expression inH2.35 cell transduced with capsid optimized AAV2 vectors. The mostefficient tyrosine, serine and threonine mutations were combined onsingle AAV2 capsid to produce several optimized AAV mutants. Efficiencyof each vector was estimated on immortalized murine hepatocytes. FIG.28A shows EGFP expression analysis at 48 hrs' post-infection at MOI of1×10³ vg/cell. FIG. 28B summarizes the quantification of transductionefficiency of each of the optimized scAAV2 vectors. *P<0.005, **P<0.001vs. WT AAV2;

FIG. 29A and FIG. 29B illustrate the kinetics of EGFP expression inH2.35 cell mediated by capsid optimized AAV vectors. FIG. 29A shows EGFPexpression analysis at 16, 24 and 48 hrs' post-infection at MOI of 1×10³vg/cell. FIG. 29B illustrates results from quantification oftransduction efficiency of each of the optimized scAAV2 vectors.*P<0.005, **P<0.001 vs. WT AAV2;

FIG. 30A and FIG. 30B show the analysis of intracellular trafficking ofAAV multiple mutant vectors to the nucleus. Nuclear and cytoplasmicfractions of H2.35 cell infected with AAV2-WT, AAV2-Y444F+Y500F+Y730F orthe AAV2-Y444F+Y500F+Y730F+T491V multi-mutant were separated and qPCRanalysis was performed to evaluate vector genome distribution withincells at 16 hrr (FIG. 30A) and 48 hrr (FIG. 30B) post infection.**P<0.001 vs. WT in nucleus was considered as significant;

FIG. 31A and FIG. 31B show the in vivo imaging of luciferase geneexpression following tail vein injection of multiple site-directed AAV2capsid mutants. C57BL/6 mice were injected with 1×10¹⁰ vg/animal ofseveral most efficient mutant scAAV vectors carrying luciferase gene.Live images were taken to analyses difference in luciferase activity.The visual output represents the number of photons emitted/second/cm² asa false color image where the maximum is red and the minimum is blue(FIG. 31A) and relative signal intensity (FIG. 31B) *P<0.005 wasconsidered as significant;

FIG. 32A and FIG. 32B illustrate the AAV2 capsid surface. FIG. 32A showsthe capsid surface of AAV2 (grey) with the 17 surface threonine residuesmutated in blue (251, 329, 330, 454, 503, 581, 592, 597, 660, 671, 701,713, 716), green (455), yellow (491), brown (550), and pink (659). Thesurface location of T329, T330, T713 and T716 are indicated by arrows.The five-fold symmetry related DE loops (between the βD and βE strands)are colored in orange. The HI loops (between the βH and βI strands) arecolored white and S662 located in this loop is in red. The white dashedtriangle in FIG. 32A depicts a viral asymmetric unit bounded by afive-fold axis and two three-fold axes with a two-fold axis between thethree-folds. Dashed ovals delineate the approximate footprints (2/60) ofthreonine residues that affect transduction when mutated. FIG. 32B showsa “roadmap” projection of the AAV2 capsid surface residues within aviral asymmetric unit. The areas covered by AAV2 surface threonines andS662 are colored as in FIG. 32A. The residues in the tyrosine triplemutant residues, 444, 500, and 730 are shown in shades of purple. Dashedovals are as described in FIG. 23A. Dashed rectangle (blue) showsresidues previously determined to be important in heparin sulfatereceptor binding for AAV2 and AAV6 (Wu et al., 2006; Opie et al., 2003);

FIG. 33A, FIG. 33B, and FIG. 33C illustrate the amino acid alignment ofthe wild-type capsids from serotypes AAV1 through AAV10. FIG. 33A showsamino acid alignment of the wild-type AAV1-10 serotype capsids (SEQ IDNO:1 through SEQ ID NO:10). FIG. 33B shows amino acid alignment of thewild-type AAV1-AAV10 serotype capsids, as well as surface-exposed serineand threonine residues that are conserved in among AAV1-AAV10 capsids(conserved, surface-exposed residues are shown in bold); and FIG. 33Cshows conserved, surface-exposed tyrosine residues in the wild-typeAAV1-AAV12 capsids, as well as embodiments of amino acid modifications.The tyrosine residues conserved among AAV1-AAV12 are shown in bold;

FIG. 34 show packaging and transduction efficiencies of variousserine→valine mutated AAV2 vectors relative to that of WT AAV2 vectorsand the amino acid alignment of wild-type AAV1-AAV10 capsids;

FIG. 35 depicts packaging and transduction efficiencies of serine-mutantvectors replaced with various samino acids relative to WT AAV2 vectors;

FIG. 36A, FIG. 36B, FIG. 36C, FIG. 36D, and FIG. 36E show transductionefficiency of rAAV3 and rAAV8 vectors in human HCC tumors in a murinexenograft model in vivo. Female and male Huh7 tumor-bearing NSG micewere used for tail-vein injection with either rAAV3-CBAp-FLuc orrAAV8-CBAp-FLuc vectors at 1×10¹¹ vgs/mouse. n=4 per group. FIG. 36Ashows representative images of mouse whole body bioluminescent images at3 days post-vector administration are shown. FIG. 36B illustrates thequantitative analysis of transgene expression data from whole bodybioluminescent images of mice at 3-days' post-vector administration.FIG. 36C shows Huh7 tumor-bearing male NSG mice were used for directintra-tumor injections with either rAAV3-CBAp-FLuc or rAAV8-CBAp-FLucvectors at a lower dose of 1×10¹¹ vgs/mouse (L) or at a higher dose of1×10¹² vgs/mouse (H). n=4 per group. Representative images of lower doseat 7 days post-vector administration are shown. FIG. 36D presents thequantitative data for transgene expression in tumors and liver from miceinjected with rAAV3 or rAAV8 vectors at 3 days or 7 days post-vectoradministration. FIG. 36E shows vector genome copy numbers persisting inthe liver tissue samples from mice injected with higher dose of rAAV3 orrAAV8 vectors 7 days post-vector administration;

FIG. 37A, FIG. 37B, FIG. 37C, FIG. 37D, FIG. 37E, and FIG. 37F showtransduction efficiency of WT and capsid-modified rAAV3 vectors in humanliver cancer cells in vitro. FIG. 37A shows Huh7 cells were transducedwith the indicated viral vectors carrying the CBAp-FLuc expressioncassette at 5,000 vgs/cell. FIG. 37B shows HepG2 cells and FIG. 37Cshows Hep293TT cells were transduced with the indicated viral vectorscarrying the CBAp-EGFP expression cassette at 5,000 vgs/cell. FIG. 37Dshows Huh7 cells were transduced with the indicated viral vectorscarrying the CBAp-EGFP expression cassette at 5,000 vgs/cell either inthe absence or presence of low (100 ng/mL) or high (100 μg/mL) doses ofsoluble heparin. FIG. 37E shows Huh7 cells were transduced with theindicated viral vectors carrying the CBAp-EGFP expression cassette at5,000 vgs/cell in either the absence or presence of 5 μg/mL hHGF. FIG.37F shows human T47D cells or T47D+hHGFR cells were transduced withindicated viral vectors carrying the CBAp-EGFP expression cassette at5,000 vgs/cell. All transgene expression levels were determined 48 hrspost-transduction;

FIG. 38A, FIG. 38B, FIG. 38C, and FIG. 38D show transduction efficiencyof exemplary rAAV3 capsid-mutated vectors in vivo. FIG. 38A shows humanHuh7- or Hep293TT liver tumor-bearing NSG mice were used for tail-veininjections with the indicated mutant viral vectors carrying theCBAp-FLuc expression cassette at 5×10¹⁰ vgs/mouse. n=4 per each group.On Day 3, mouse whole-body bioluminescent images were obtained, followedby dissection of both growing tumor and normal liver. Representativeimages are shown. FIG. 38B shows quantitative data showing FLucexpression in Huh7- and Hep293TT-derived tumors. Vector genome copynumbers are shown in Huh7 tumors (FIG. 38C), and in normal livers (FIG.38D);

FIG. 39A, FIG. 39B and FIG. 39C show exemplary embodiments of thepresent invention. FIG. 39A shows suppression of human livertumorigenesis in a murine xenograft model by optimized rAAV3 vectorsexpressing the TCS gene; FIG. 39B and FIG. 39C show results of Huh7-FLuctumor-bearing mice were used for tail-vein injections with the indicatedviral vectors at 5×10¹⁰ vgs/mouse at Day 0, and tumor growth wasmonitored over time until Day 11. FIG. 39B depicts representativewhole-body bioluminescence images of mice from both groups at Day 8.FIG. 39C summarizes serum activities of AST and ALT that were measuredin rAAV3-TCS vector-injected and rAAV3-EGFP vector-injected mice at Day11 by spectrophotometric methods. Data are presented as mean±SD.(n=5/group);

FIG. 40A, FIG. 40B and FIG. 40C show exemplary vector constructs andpolynucleotide sequences useful in accordance with one aspect of thepresent invention. FIG. 40A shows the schematic structures of the rAAVvectors used in these studies. HP: hairpin; D: D-sequence in the AAVinverted terminal repeat (ITR); CBAp: CMV enhancer/chicken β-actinpromoter; FLuc: firefly luciferase; hGH (A)n: human growth hormone polyAsequence; HP-: hairpin structure without the terminal resolution site(trs); EGFP: enhanced green fluorescence protein; AFPp: humanα-fetoprotein promoter; TCS: Tricosanthin; FIG. 40B shows the nucleotidesequence of the original TCS gene. The start codon (ATG) and the stopcodon (TAG) are shown in green and red fonts, respectively. FIG. 40Cshows the nucleotide sequence of the FLAG-tagged TCS gene. The EcoRI andXhoI restriction enzyme sites used for cloning the chemicallysynthesized TCS gene are shown in bold italic font, respectively, andthe FLAG-tag sequence containing the stop codon (TAA) is underlined; and

FIG. 41 shows the transduction efficiency of various single-, double-,and multiple-mutant scAAV3-CBAp-EGFP vectors in human HCC cell line,Huh7.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 1 (AAV1);

SEQ ID NO:2 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 2 (AAV2);

SEQ ID NO:3 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 3 (AAV3);

SEQ ID NO:4 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 4 (AAV4);

SEQ ID NO:5 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 5 (AAV5);

SEQ ID NO:6 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 6 (AAV6);

SEQ ID NO:7 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 7 (AAV7);

SEQ ID NO:8 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 8 (AAV8);

SEQ ID NO:9 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 9 (AAV9);

SEQ ID NO:10 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 10 (AAV10);

SEQ ID NO:11 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:12 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:13 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:14 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:15 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:16 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:17 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:18 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:19 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:20 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:21 is an oligonucleotide primer sequence useful according tothe present invention;

SEQ ID NO:22 is a nucleic acid sequence containing the putatitve bindingsite for NF-kB-responsive transcription factors (See FIG. 5);

SEQ ID NO:23 is a single-stranded nucleic acid sequence probe (see FIG.10);

SEQ ID NO:24 is a double-stranded nucleic acid sequence probe (see FIG.10);

SEQ ID NO:25 is a single-stranded nucleic acid sequence probe (see FIG.10);

SEQ ID NO:26 is the nucleic acid sequence of the TCS gene (see FIG.40B); and

SEQ ID NO:27 is the nucleic acid sequence of the FLAG-tagged TCS gene(see FIG. 40C).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Illustrative embodiments of the invention are described below. In theinterest of clarity, not all features of an actual implementation aredescribed in this specification. It will of course be appreciated thatin the development of any such actual embodiment, numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, such as compliance with system-related andbusiness-related constraints, which will vary from one implementation toanother. Moreover, it will be appreciated that such a development effortmight be complex and time-consuming, but would be a routine undertakingfor those of ordinary skill in the art having the benefit of thisdisclosure.

Recombinant adeno-associated virus (AAV) vectors have been usedsuccessfully for in vivo gene transfer in numerous pre-clinical animalmodels of human disease, and have been used successfully for long-termexpression of a wide variety of therapeutic genes (Daya and Berns, 2008;Niemeyer et al., 2009; Owen et al., 2002; Keen-Rhinehart et al., 2005;Scallan et al., 2003; Song et al., 2004). AAV vectors have alsogenerated long-term clinical benefit in humans when targeted toimmune-privileged sites, i.e., ocular delivery for Leber's congenitalamaurosis (Bainbridge et al., 2008; Maguire et al., 2008; Cideciyan etal., 2008). A major advantage of this vector is its comparatively lowimmune profile, eliciting only limited inflammatory responses and, insome cases, even directing immune tolerance to transgene products(LoDuca et al., 2009). Nonetheless, the therapeutic efficiency, whentargeted to non-immune privileged organs, has been limited in humans dueto antibody and CD8⁺ T cell responses against the viral capsid, while inanimal models, adaptive responses to the transgene product have alsobeen reported (Manno et al., 2006; Mingozzi et al., 2007; Muruve et al.,2008; Vandenberghe and Wilson, 2007; Mingozzi and High, 2007). Theseresults suggested that immune responses remain a concern for AAVvector-mediated gene transfer.

Based on pre-clinical data from murine models (Snyder et al., 1999), AAVwas considered as minimally immunogenic for years, due to absence ofprior exposure of these antigens in these models and the presence ofvariety of tolerance-inducing mechanisms against the vector (Dobrzynskiet al., 2004; Cao et al., 2007). This was best illustrated in genetransfer studies in murine and canine models of hemophilia B, whichshowed remarkable therapeutic efficiency (5-25% of F.IX levels) andlong-term (2-8 years) and stable F.IX expression (Snyder et al., 1999).In the first clinical trial using AAV to deliver the human F.IX gene tothe liver in subjects with hemophilia B, therapeutic levels (˜11.8%) ofF.IX expression were observed at a high dose of vector (2×10¹² vgs/kgbody weight) (Manno et al., 2006).

However, 4-6 weeks after gene transfer, an AAV capsid-specific T cellresponse was observed that coincided with a rise in liver transaminasesand a drop in F.IX transgene expression to baseline levels. This CD8⁺ Tcell-mediated immune response was unexpected (Mingozzi et al., 2007), asthis had not been observed in any pre-clinical animal models. This studyand several others have implicated the host inflammatory and innateimmune responses for cytotoxic T-lymphocyte mediated elimination oftransduced hepatocytes (Zhu et al., 2009; Li et al., 2009; Madsen etal., 2009). Subsequently, a great deal of effort has been devoted tocircumvent the host immune response to AAV vectors. These include theuse of alternate naturally occurring AAV serotypes such as AAV1 (Brantlyet al., 2009; Cohn et al., 2007) or AAV8 (Nathwani et al., 2006), theuse of shuffled capsids (Gray et al., 2010), or surface-exposedtyrosine-mutant AAV2 (Markusic et al., 2010) vectors. In addition,strategies to counter the risks associated with the immune response haveincluded the use of transgene constructs which have targeted expressionin the host tissue (Wang et al., 2010), or the development of transientimmune-suppression protocols (Jiang et al., 2006).

Although such strategies have incrementally improved the safety of AAVgene transfer, their efficacy in humans remains to be seen. For example,immune suppression with cyclosporine and MMF was effective at lower AAV1vector dose (3×10¹¹ vg/kg) but failed to prevent IFN-α CD8+ T cellresponses against capsid at high doses (1×10¹² vg/kg) duringmuscle-directed gene transfer in patients with lipoprotein lipasedeficiency (Ross et al., 2006). These data underscore the importance ofpursuing further studies on the biology of the virus-host cellinteractions to identify the first “danger signal” in response to AAVinfection. It was reasoned that understanding how the potential activityand the selectivity of proteins associated with inflammatory and innateimmune response are regulated in host cells upon transduction with AAVmight offer clues to address obstacles of the host immune responseagainst the capsid and/or the transgene product. Although compared withother viral vectors, AAV vectors are inefficient in transducingprofessional APCs such as DCs, additional signals that activate NF-κBwould lead to increased transgene expression in these cells, therebyincreasing the risk of adaptive responses to the transgene product.

Recombinant vectors based on AAV serotype 2 are currently in use in anumber of gene therapy clinical trials (Daya and Berns, 2008), and haverecently shown remarkable efficacy in the treatment of Leber'scongenital amaurosis (Bainbridge et al., 2008; Cideciyan et al., 2008;Maguire et al., 2008). However, concerns have been raised with referenceto the humoral response to AAV2 vectors based on the high prevalence ofsero-positivity in the general population (˜80 to 90%) (Boutin et al.,2008; Mendell et al., 2010; Manno et al., 2006). The discovery of manynovel AAV serotypes has prompted the development of AAV vectors tocircumvent this potential problem (Muramatsu et al., 1996; Chiorini etal., 1997; Chiorini et al., 199; Rutledge et al., 1998; Gao et al.,2002; Gao et al., 2004).

For example, recombinant AAV8 vectors were recently reported to betherapeutic in a mouse model of liver cancer (Kato et al., 2006).However, several groups have described various strategies to targethuman liver cancer cells in murine models using AAV2 vectors (Su et al.,1996; Peng et al., 2000; Su et al., 2000; Ma et al., 2005; Wang et al.,2005; Tse et al., 2008; Zhang et al., 2008; Malecki et al., 2009; Wanget al., 2010). To identify the most efficient AAV serotype to targethuman liver cancer cells, three different human liver cancer cell lineswere shown to be transduced extremely efficiently by AAV3 vectors(Glushakova et al., 2009). Human hepatocyte growth factor receptor(hHGFR) was subsequently identified as a cellular co-receptor for AAV3infection (Ling et al., 2010). The precise role of hHGFR, especially therole of tyrosine kinase activity associated with the intracellulardomain of hHGFR, in AAV3-mediated transduction initially remainedunclear. Data in Example 5 (see below) provided a more-detailedexplanation of AAV3-hHGFR interactions, and illustrated the developmentof optimized capsid-mutated AAV3 vectors for use in targeting humanliver cancer cells.

rAAV Capsid Proteins

Supramolecular assembly of 60 individual capsid protein subunits into anon-enveloped, T-1 icosahedral lattice capable of protecting a 4.7-kbsingle-stranded DNA genome is a critical step in the life-cycle of thehelper-dependent human parvovirus, adeno-associated virus2 (AAV2). Themature 20-nm diameter AAV2 particle is composed of three structuralproteins designated VP1, VP2, and VP3 (molecular masses of 87, 73, and62 kDa respectively) in a ratio of 1:1:18. Based on its symmetry andthese molecular weight estimates, of the 60 capsid proteins comprisingthe particle, three are VP1 proteins, three are VP2 proteins, andfifty-four are VP3 proteins. The employment of three structural proteinsmakes AAV serotypes unique among parvoviruses, as all others knownpackage their genomes within icosahedral particles composed of only twocapsid proteins. The anti-parallel β-strand barreloid arrangement ofthese 60 capsid proteins results in a particle with a defined tropismthat is highly resistant to degradation. Modification of one or moretyrosine residues in one or more of the capsid proteins has been shownby the inventors to achieve superior transfection at lower dose andlower cost than conventional protocols. By site-specifically modifyingone or more tyrosine residues on the surface of the capsid, theinventors have achieved significant improvement in transductionefficiency.

Uses for Improved, Capsid-Modified rAAV Vectors

The present invention provides compositions including one or more of thedisclosed tyrosine-modified rAAV vectors comprised within a kit fordiagnosing, preventing, treating or ameliorating one or more symptoms ofa mammalian disease, injury, disorder, trauma or dysfunction. Such kitsmay be useful in diagnosis, prophylaxis, and/or therapy, andparticularly useful in the treatment, prevention, and/or amelioration ofone or more symptoms of cancer, diabetes, autoimmune disease, kidneydisease, cardiovascular disease, pancreatic disease, intestinal disease,liver disease, neurological disease, neuromuscular disorder, neuromotordeficit, neuroskeletal impairment, neurological disability, neurosensorydysfunction, stroke, ischemia, eating disorder, α₁-antitrypsin (AAT)deficiency, Batten's disease, Alzheimer's disease, sickle cell disease,β-thalassamia, Huntington's disease, Parkinson's disease, skeletaldisease, trauma, pulmonary disease, or any combination thereof.

The invention also provides for the use of a composition disclosedherein in the manufacture of a medicament for treating, preventing orameliorating the symptoms of a disease, disorder, dysfunction, injury ortrauma, including, but not limited to, the treatment, prevention, and/orprophylaxis of a disease, disorder or dysfunction, and/or theamelioration of one or more symptoms of such a disease, disorder ordysfunction. Exemplary conditions for which rAAV viral based genetherapy may find particular utility include, but are not limited to,cancer, diabetes, sickle cell disease, β-thalassamia, autoimmunedisease, kidney disease, cardiovascular disease, pancreatic disease,diseases of the eye, intestinal disease, liver disease, neurologicaldisease, neuromuscular disorder, neuromotor deficit, neuroskeletalimpairment, neurological disability, neurosensory dysfunction, stroke,α₁-antitrypsin (AAT) deficiency, Batten's disease, ischemia, an eatingdisorder, Alzheimer's disease, Huntington's disease, Parkinson'sdisease, skeletal disease, pulmonary disease, and any combinationsthereof.

The invention also provides a method for treating or ameliorating thesymptoms of such a disease, injury, disorder, or dysfunction in amammal. Such methods generally involve at least the step ofadministering to a mammal in need thereof, one or more of thetyrosine-modified rAAV vectors as disclosed herein, in an amount and fora time sufficient to treat or ameliorate the symptoms of such a disease,injury, disorder, or dysfunction in the mammal.

Such treatment regimens are particularly contemplated in human therapy,via administration of one or more compositions either intramuscularly,intravenously, subcutaneously, intrathecally, intraperitoneally, or bydirect injection into an organ or a tissue of the mammal under care.

The invention also provides a method for providing to a mammal in needthereof, a therapeutically-effective amount of the rAAV compositions ofthe present invention, in an amount, and for a time effective to providethe patient with a therapeutically-effective amount of the desiredtherapeutic agent(s) encoded by one or more nucleic acid segmentscomprised within the rAAV vector. Preferably, the therapeutic agent isselected from the group consisting of a polypeptide, a peptide, anantibody, an antigen-binding fragment, a ribozyme, a peptide nucleicacid, an siRNA, an RNAi, an antisense oligonucleotide, an antisensepolynucleotide, a diagnostic marker, a diagnostic molecule, a reportermolecule, and any combination thereof.

AAV Vector Compositions

One important aspect of the present methodology is the fact that theimproved rAAV vectors described herein permit the delivery of smallertiters of viral particles in order to achieve the same transductionefficiency as that obtained using higher levels of conventional,non-surface capsid modified rAAV vectors. To that end, the amount of AAVcompositions and time of administration of such compositions will bewithin the purview of the skilled artisan having benefit of the presentteachings. In fact, the inventors contemplate that the administration oftherapeutically-effective amounts of the disclosed compositions may beachieved by a single administration, such as for example, a singleinjection of sufficient numbers of infectious particles to providetherapeutic benefit to the patient undergoing such treatment.Alternatively, in some circumstances, it may be desirable to providemultiple, or successive administrations of the AAV vector compositions,either over a relatively short, or over a relatively prolonged period,as may be determined by the medical practitioner overseeing theadministration of such compositions. For example, the number ofinfectious particles administered to a mammal may be approximately 10⁷,10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or even higher, infectiousparticles/mL, given either as a single dose (or divided into two or moreadministrations, etc.) as may be required to achieve therapy of theparticular disease or disorder being treated. In fact, in certainembodiments, it may be desirable to administer two or more differentrAAV vector-based compositions, either alone, or in combination with oneor more other diagnostic agents, drugs, bioactives, or such like, toachieve the desired effects of a particular regimen or therapy. In mostrAAV-vectored, gene therapy-based regimens, the inventors contemplatethat lower titers of infectious particles will be required when usingthe modified-capsid rAAV vectors described herein, as compared to theuse of equivalent wild-type, or corresponding “un-modified” rAAVvectors.

As used herein, the terms “engineered” and “recombinant” cells areintended to refer to a cell into which an exogenous polynucleotidesegment (such as DNA segment that leads to the transcription of abiologically active molecule) has been introduced. Therefore, engineeredcells are distinguishable from naturally occurring cells, which do notcontain a recombinantly introduced exogenous DNA segment. Engineeredcells are, therefore, cells that comprise at least one or moreheterologous polynucleotide segments introduced through the hand of man.

To express a therapeutic agent in accordance with the present inventionone may prepare a tyrosine-modified rAAV expression vector thatcomprises a therapeutic agent-encoding nucleic acid segment under thecontrol of one or more promoters. To bring a sequence “under the controlof” a promoter, one positions the 5′ end of the transcription initiationsite of the transcriptional reading frame generally between about 1 andabout 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter.The “upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded polypeptide. This is the meaning of“recombinant expression” in this context. Particularly preferredrecombinant vector constructs are those that comprise an rAAV vector.Such vectors are described in detail herein.

When the use of such vectors is contemplated for introduction of one ormore exogenous proteins, polypeptides, peptides, ribozymes, and/orantisense oligonucleotides, to a particular cell transfected with thevector, one may employ the capsid-modified rAAV vectors disclosed hereinto deliver one or more exogenous polynucleotides to a selected hostcell.

Pharmaceutical Compositions

The genetic constructs of the present invention may be prepared in avariety of compositions, and may also be formulated in appropriatepharmaceutical vehicles for administration to human or animal subjects.The rAAV molecules of the present invention and compositions comprisingthem provide new and useful therapeutics for the treatment, control, andamelioration of symptoms of a variety of disorders, and in particular,articular diseases, disorders, and dysfunctions, including for exampleosteoarthritis, rheumatoid arthritis, and related disorders.

The invention also provides compositions comprising one or more of thedisclosed capsid-modified rAAV vectors, expression systems, virions,viral particles, mammalian cells, or combinations thereof. In certainembodiments, the present invention provides pharmaceutical formulationsof one or more capsid-modified rAAV vectors disclosed herein foradministration to a cell or an animal, either alone or in combinationwith one or more other modalities of therapy, and in particular, fortherapy of human cells, tissues, and diseases affecting man. Formulationof pharmaceutically-acceptable excipients and carrier solutions iswell-known to those of skill in the art, as is the development ofsuitable dosing and treatment regimens for using the particularcompositions described herein in a variety of treatment regimens,including e.g., oral, parenteral, intravenous, intranasal,intra-articular, intramuscular administration and formulation.

EXEMPLARY DEFINITIONS

In accordance with the present invention, polynucleotides, nucleic acidsegments, nucleic acid sequences, and the like, include, but are notlimited to, DNAs (including and not limited to genomic or extragenomicDNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but notlimited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleicacid segments either obtained from natural sources, chemicallysynthesized, modified, or otherwise prepared or synthesized in whole orin part by the hand of man.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defined below:

In accordance with long standing patent law convention, the words “a”and “an” when used in this application, including the claims, denote“one or more.”

The terms “about” and “approximately” as used herein, areinterchangeable, and should generally be understood to refer to a rangeof numbers around a given number, as well as to all numbers in a recitedrange of numbers (e.g., “about 5 to 15” means “about 5 to about 15”unless otherwise stated). Moreover, all numerical ranges herein shouldbe understood to include each whole integer within the range.

As used herein, the term “carrier” is intended to include anysolvent(s), dispersion medium, coating(s), diluent(s), buffer(s),isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s) orsuch like, or a combination thereof, that is pharmaceutically acceptablefor administration to the relevant animal. The use of one or moredelivery vehicles for chemical compounds in general, andchemotherapeutics in particular, is well known to those of ordinaryskill in the pharmaceutical arts. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe diagnostic, prophylactic, and therapeutic compositions iscontemplated. One or more supplementary active ingredient(s) may also beincorporated into, or administered in association with, one or more ofthe disclosed chemotherapeutic compositions.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment obtained from a biological sample using one ofthe compositions disclosed herein refers to one or more DNA segmentsthat have been isolated away from, or purified free from, total genomicDNA of the particular species from which they are obtained. Includedwithin the term “DNA segment,” are DNA segments and smaller fragments ofsuch segments, as well as recombinant vectors, including, for example,plasmids, cosmids, phage, viruses, and the like.

The term “effective amount,” as used herein, refers to an amount that iscapable of treating or ameliorating a disease or condition or otherwisecapable of producing an intended therapeutic effect. The term “forexample” or “e.g.,” as used herein, is used merely by way of example,without limitation intended, and should not be construed as referringonly those items explicitly enumerated in the specification.

As used herein, a “heterologous” is defined in relation to apredetermined referenced gene sequence. For example, with respect to astructural gene sequence, a heterologous promoter is defined as apromoter which does not naturally occur adjacent to the referencedstructural gene, but which is positioned by laboratory manipulation.Likewise, a heterologous gene or nucleic acid segment is defined as agene or segment that does not naturally occur adjacent to the referencedpromoter and/or enhancer elements.

As used herein, the term “homology” refers to a degree ofcomplementarity between two or more polynucleotide or polypeptidesequences. The word “identity” may substitute for the word “homology”when a first nucleic acid or amino acid sequence has the exact sameprimary sequence as a second nucleic acid or amino acid sequence.Sequence homology and sequence identity can be determined by analyzingtwo or more sequences using algorithms and computer programs known inthe art. Such methods may be used to assess whether a given sequence isidentical or homologous to another selected sequence.

As used herein, “homologous” means, when referring to polynucleotides,sequences that have the same essential nucleotide sequence, despitearising from different origins. Typically, homologous nucleic acidsequences are derived from closely related genes or organisms possessingone or more substantially similar genomic sequences. By contrast, an“analogous” polynucleotide is one that shares the same function with apolynucleotide from a different species or organism, but may have asignificantly different primary nucleotide sequence that encodes one ormore proteins or polypeptides that accomplish similar functions orpossess similar biological activity. Analogous polynucleotides may oftenbe derived from two or more organisms that are not closely related(e.g., either genetically or phylogenetically).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of ordinary skill) or by visualinspection.

As used herein, the phrase “in need of treatment” refers to a judgmentmade by a caregiver such as a physician or veterinarian that a patientrequires (or will benefit in one or more ways) from treatment. Suchjudgment may made based on a variety of factors that are in the realm ofa caregiver's expertise, and may include the knowledge that the patientis ill as the result of a disease state that is treatable by one or morecompound or pharmaceutical compositions such as those set forth herein.

As used herein, the term “nucleic acid” includes one or more types of:polydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other type ofpolynucleotide that is an N-glycoside of a purine or pyrimidine base, ormodified purine or pyrimidine bases (including abasic sites). The term“nucleic acid,” as used herein, also includes polymers ofribonucleosides or deoxyribonucleosides that are covalently bonded,typically by phosphodiester linkages between subunits, but in some casesby phosphorothioates, methylphosphonates, and the like. “Nucleic acids”include single- and double-stranded DNA, as well as single- anddouble-stranded RNA. Exemplary nucleic acids include, withoutlimitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), smallinterfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA(snRNA), and small temporal RNA (stRNA), and the like, and anycombination thereof.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by the hand of man in alaboratory is naturally-occurring. As used herein, laboratory strains ofrodents that may have been selectively bred according to classicalgenetics are considered naturally occurring animals.

The term “operably linked,” as used herein, refers to that the nucleicacid sequences being linked are typically contiguous, or substantiallycontiguous, and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

As used herein, the term “patient” (also interchangeably referred to as“host” or “subject”) refers to any host that can receive one or more ofthe pharmaceutical compositions disclosed herein. Preferably, thesubject is a vertebrate animal, which is intended to denote any animalspecies (and preferably, a mammalian species such as a human being). Incertain embodiments, a “patient” refers to any animal host includingwithout limitation any mammalian host. Preferably, the term refers toany mammalian host, the latter including but not limited to, human andnon-human primates, bovines, canines, caprines, cavines, corvines,epines, equines, felines, hircines, lapines, leporines, lupines,murines, ovines, porcines, ranines, racines, vulpines, and the like,including livestock, zoological specimens, exotics, as well as companionanimals, pets, and any animal under the care of a veterinarypractitioner. A patient can be of any age at which the patient is ableto respond to inoculation with the present vaccine by generating animmune response. In particular embodiments, the mammalian patient ispreferably human.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that preferably do not produce an allergic or similaruntoward reaction when administered to a mammal, and in particular, whenadministered to a human. As used herein, “pharmaceutically acceptablesalt” refers to a salt that preferably retains the desired biologicalactivity of the parent compound and does not impart any undesiredtoxicological effects. Examples of such salts include, withoutlimitation, acid addition salts formed with inorganic acids (e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid, and the like); and salts formed with organic acidsincluding, without limitation, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic)acid, alginic acid, naphthoic acid, polyglutamic acid,naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonicacid; salts with polyvalent metal cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium,and the like; salts formed with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; and combinationsthereof.

The term “pharmaceutically acceptable salt” as used herein refers to acompound of the present disclosure derived from pharmaceuticallyacceptable bases, inorganic or organic acids. Examples of suitable acidsinclude, but are not limited to, hydrochloric, hydrobromic, sulfuric,nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic,salicyclic, succinic, toluene-p-sulfonic, tartaric, acetic, citric,methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,trifluoroacetic and benzenesulfonic acids. Salts derived fromappropriate bases include, but are not limited to, alkali such as sodiumand ammonia.

As used herein, the terms “prevent,” “preventing,” “prevention,”“suppress,” “suppressing,” and “suppression” as used herein refer toadministering a compound either alone or as contained in apharmaceutical composition prior to the onset of clinical symptoms of adisease state so as to prevent any symptom, aspect or characteristic ofthe disease state. Such preventing and suppressing need not be absoluteto be deemed medically useful.

The term “promoter,” as used herein refers to a region or regions of anucleic acid sequence that regulates transcription.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and includesany chain or chains of two or more amino acids. Thus, as used herein,terms including, but not limited to “peptide,” “dipeptide,”“tripeptide,” “protein,” “enzyme,” “amino acid chain,” and “contiguousamino acid sequence” are all encompassed within the definition of a“polypeptide,” and the term “polypeptide” can be used instead of, orinterchangeably with, any of these terms. The term further includespolypeptides that have undergone one or more post-translationalmodification(s), including for example, but not limited to,glycosylation, acetylation, phosphorylation, amidation, derivatization,proteolytic cleavage, post-translation processing, or modification byinclusion of one or more non-naturally occurring amino acids.Conventional nomenclature exists in the art for polynucleotide andpolypeptide structures. For example, one-letter and three-letterabbreviations are widely employed to describe amino acids: Alanine (A;Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp),Cysteine (C; Cys), Glutamine (Q; Gln), Glutamic Acid (E; Glu), Glycine(G; Gly), Histidine (H; His), Isoleucine (I; Ile), Leucine (L; Leu),Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine(S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr),Valine (V; Val), and Lysine (K; Lys). Amino acid residues describedherein are preferred to be in the “l” isomeric form. However, residuesin the “d” isomeric form may be substituted for any l-amino acid residueprovided the desired properties of the polypeptide are retained.

“Protein” is used herein interchangeably with “peptide” and“polypeptide,” and includes both peptides and polypeptides producedsynthetically, recombinantly, or in vitro and peptides and polypeptidesexpressed in vivo after nucleic acid sequences are administered into ahost animal or human subject. The term “polypeptide” is preferablyintended to refer to any amino acid chain length, including those ofshort peptides from about 2 to about 20 amino acid residues in length,oligopeptides from about 10 to about 100 amino acid residues in length,and longer polypeptides including from about 100 amino acid residues ormore in length. Furthermore, the term is also intended to includeenzymes, i.e., functional biomolecules including at least one amino acidpolymer. Polypeptides and proteins of the present invention also includepolypeptides and proteins that are or have been post-translationallymodified, and include any sugar or other derivative(s) or conjugate(s)added to the backbone amino acid chain.

The term “recombinant” indicates that the material (e.g., apolynucleotide or a polypeptide) has been artificially or synthetically(non-naturally) altered by human intervention. The alteration can beperformed on the material within or removed from, its naturalenvironment or state. Specifically, e.g., a promoter sequence is“recombinant” when it is produced by the expression of a nucleic acidsegment engineered by the hand of man. For example, a “recombinantnucleic acid” is one that is made by recombining nucleic acids, e.g.,during cloning, DNA shuffling or other procedures, or by chemical orother mutagenesis; a “recombinant polypeptide” or “recombinant protein”is a polypeptide or protein which is produced by expression of arecombinant nucleic acid; and a “recombinant virus,” e.g., a recombinantAAV virus, is produced by the expression of a recombinant nucleic acid.

The term “regulatory element,” as used herein, refers to a region orregions of a nucleic acid sequence that regulates transcription.Exemplary regulatory elements include, but are not limited to,enhancers, post-transcriptional elements, transcriptional controlsequences, and such like.

The term “RNA segment” refers to an RNA molecule that has been isolatedfree of total cellular RNA of a particular species. Therefore, RNAsegments can refer to one or more RNA segments (either of native orsynthetic origin) that have been isolated away from, or purified freefrom, other RNAs. Included within the term “RNA segment,” are RNAsegments and smaller fragments of such segments.

The term “substantially corresponds to,” “substantially homologous,” or“substantial identity,” as used herein, denote a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably, atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared.

The percentage of sequence identity may be calculated over the entirelength of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides.

When highly-homologous fragments are desired, the extent of percentidentity between the two sequences will be at least about 80%,preferably at least about 85%, and more preferably about 90% or 95% orhigher, as readily determined by one or more of the sequence comparisonalgorithms well-known to those of skill in the art, such as e.g., theFASTA program analysis described by Pearson and Lipman (1988). The term“subject,” as used herein, describes an organism, including mammals suchas primates, to which treatment with the compositions according to thepresent invention can be provided. Mammalian species that can benefitfrom the disclosed methods of treatment include, but are not limited to,apes; chimpanzees; orangutans; humans; monkeys; domesticated animalssuch as dogs and cats; livestock such as horses, cattle, pigs, sheep,goats, and chickens; and other animals such as mice, rats, guinea pigs,and hamsters.

As used herein, the term “structural gene” is intended to generallydescribe a polynucleotide, such as a gene, that is expressed to producean encoded peptide, polypeptide, protein, ribozyme, catalytic RNAmolecule, or antisense molecule.

The term “subject,” as used herein, describes an organism, includingmammals such as primates, to which treatment with the compositionsaccording to the present invention can be provided. Mammalian speciesthat can benefit from the disclosed methods of treatment include, butare not limited to, humans, non-human primates such as apes;chimpanzees; monkeys, and orangutans, domesticated animals, includingdogs and cats, as well as livestock such as horses, cattle, pigs, sheep,and goats, or other mammalian species including, without limitation,mice, rats, guinea pigs, rabbits, hamsters, and the like.

“Transcriptional regulatory element” refers to a polynucleotide sequencethat activates transcription alone or in combination with one or moreother nucleic acid sequences. A transcriptional regulatory element can,for example, comprise one or more promoters, one or more responseelements, one or more negative regulatory elements, and/or one or moreenhancers.

As used herein, a “transcription factor recognition site” and a“transcription factor binding site” refer to a polynucleotidesequence(s) or sequence motif(s) which are identified as being sites forthe sequence-specific interaction of one or more transcription factors,frequently taking the form of direct protein-DNA binding. Typically,transcription factor binding sites can be identified by DNAfootprinting, gel mobility shift assays, and the like, and/or can bepredicted on the basis of known consensus sequence motifs, or by othermethods known to those of skill in the art.

“Transcriptional unit” refers to a polynucleotide sequence thatcomprises at least a first structural gene operably linked to at least afirst cis-acting promoter sequence and optionally linked operably to oneor more other cis-acting nucleic acid sequences necessary for efficienttranscription of the structural gene sequences, and at least a firstdistal regulatory element as may be required for the appropriatetissue-specific and developmental transcription of the structural genesequence operably positioned under the control of the promoter and/orenhancer elements, as well as any additional cis sequences that arenecessary for efficient transcription and translation (e.g.,polyadenylation site(s), mRNA stability controlling sequence(s), etc.

As used herein, the term “transformed cell” is intended to mean a hostcell whose nucleic acid complement has been altered by the introductionof one or more exogenous polynucleotides into that cell.

As used herein, the term “transformation” is intended to generallydescribe a process of introducing an exogenous polynucleotide sequence(e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule)into a host cell or protoplast in which the exogenous polynucleotide isincorporated into at least a first chromosome or is capable ofautonomous replication within the transformed host cell. Transfection,electroporation, and “naked” nucleic acid uptake all represent examplesof techniques used to transform a host cell with one or morepolynucleotides.

As used herein, the terms “treat,” “treating,” and “treatment” refer tothe administration of one or more compounds (either alone or ascontained in one or more pharmaceutical compositions) after the onset ofclinical symptoms of a disease state so as to reduce, or eliminate anysymptom, aspect or characteristic of the disease state. Such treatingneed not be absolute to be deemed medically useful. As such, the terms“treatment,” “treat,” “treated,” or “treating” may refer to therapy, orto the amelioration or the reduction, in the extent or severity ofdisease, of one or more symptom thereof, whether before or after itsdevelopment afflicts a patient.

The phrases “isolated” or “biologically pure” refer to material that issubstantially, or essentially, free from components that normallyaccompany the material as it is found in its native state. Thus,isolated polynucleotides in accordance with the invention preferably donot contain materials normally associated with those polynucleotides intheir natural, or in situ, environment.

“Link” or “join” refers to any method known in the art for functionallyconnecting one or more proteins, peptides, nucleic acids, orpolynucleotides, including, without limitation, recombinant fusion,covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding,electrostatic bonding, and the like.

As used herein, the term “plasmid” or “vector” refers to a geneticconstruct that is composed of genetic material (i.e., nucleic acids).Typically, a plasmid or a vector contains an origin of replication thatis functional in bacterial host cells, e.g., Escherichia coli, andselectable markers for detecting bacterial host cells including theplasmid. Plasmids and vectors of the present invention may include oneor more genetic elements as described herein arranged such that aninserted coding sequence can be transcribed and translated in a suitableexpression cells. In addition, the plasmid or vector may include one ormore nucleic acid segments, genes, promoters, enhancers, activators,multiple cloning regions, or any combination thereof, including segmentsthat are obtained from or derived from one or more natural and/orartificial sources.

The term “a sequence essentially as set forth in SEQ ID NO:X” means thatthe sequence substantially corresponds to a portion of SEQ ID NO:X andhas relatively few nucleotides (or amino acids in the case ofpolypeptide sequences) that are not identical to, or a biologicallyfunctional equivalent of, the nucleotides (or amino acids) of SEQ IDNO:X. The term “biologically functional equivalent” is well understoodin the art, and is further defined in detail herein. Accordingly,sequences that have about 85% to about 90%; or more preferably, about91% to about 95%; or even more preferably, about 96% to about 99%; ofnucleotides that are identical or functionally equivalent to one or moreof the nucleotide sequences provided herein are particularlycontemplated to be useful in the practice of the invention.

Suitable standard hybridization conditions for the present inventioninclude, for example, hybridization in 50% formamide, 5×Denhardt'ssolution, 5×SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg/ml ofdenatured salmon sperm DNA at 42° C. for 16 h followed by 1 hrsequential washes with 0.1×SSC, 0.1% SDS solution at 60° C. to removethe desired amount of background signal. Lower stringency hybridizationconditions for the present invention include, for example, hybridizationin 35% formamide, 5×Denhardt's solution, 5×SSC, 25 mM sodium phosphate,0.1% SDS and 100 μg/ml denatured salmon sperm DNA or E. coli DNA at 42°C. for 16 h followed by sequential washes with 0.8×SSC, 0.1% SDS at 55°C. Those of skill in the art will recognize that conditions can bereadily adjusted to obtain the desired level of stringency.

Naturally, the present invention also encompasses nucleic acid segmentsthat are complementary, essentially complementary, and/or substantiallycomplementary to at least one or more of the specific nucleotidesequences specifically set forth herein. Nucleic acid sequences that are“complementary” are those that are capable of base-pairing according tothe standard Watson-Crick complementarity rules. As used herein, theterm “complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to one or more of the specific nucleic acid segmentsdisclosed herein under relatively stringent conditions such as thosedescribed immediately above.

As described above, the probes and primers of the present invention maybe of any length. By assigning numeric values to a sequence, forexample, the first residue is 1, the second residue is 2, etc., analgorithm defining all probes or primers contained within a givensequence can be proposed:

n to n+y, where n is an integer from 1 to the last number of thesequence and y is the length of the probe or primer minus one, where n+ydoes not exceed the last number of the sequence. Thus, for a 25-basepairprobe or primer (i.e., a “25-mer”), the collection of probes or primerscorrespond to bases 1 to 25, bases 2 to 26, bases 3 to 27, bases 4 to28, and so on over the entire length of the sequence. Similarly, for a35-basepair probe or primer (i.e., a “35-mer), exemplary primer or probesequence include, without limitation, sequences corresponding to bases 1to 35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over theentire length of the sequence. Likewise, for 40-mers, such probes orprimers may correspond to the nucleotides from the first basepair to bp40, from the second by of the sequence to bp 41, from the third bp to bp42, and so forth, while for 50-mers, such probes or primers maycorrespond to a nucleotide sequence extending from bp 1 to bp 50, frombp 2 to bp 51, from bp 3 to bp 52, from bp 4 to bp 53, and so forth.

In certain embodiments, it will be advantageous to employ one or morenucleic acid segments of the present invention in combination with anappropriate detectable marker (i.e., a “label,”), such as in the case ofemploying labeled polynucleotide probes in determining the presence of agiven target sequence in a hybridization assay. A wide variety ofappropriate indicator compounds and compositions are known in the artfor labeling oligonucleotide probes, including, without limitation,fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, etc., which are capable of being detected in a suitableassay. In particular embodiments, one may also employ one or morefluorescent labels or an enzyme tag such as urease, alkaline phosphataseor peroxidase, instead of radioactive or other environmentallyless-desirable reagents. In the case of enzyme tags, colorimetric,chromogenic, or fluorigenic indicator substrates are known that can beemployed to provide a method for detecting the sample that is visible tothe human eye, or by analytical methods such as scintigraphy,fluorimetry, spectrophotometry, and the like, to identify specifichybridization with samples containing one or more complementary orsubstantially complementary nucleic acid sequences. In the case ofso-called “multiplexing” assays, where two or more labeled probes aredetected either simultaneously or sequentially, it may be desirable tolabel a first oligonucleotide probe with a first label having a firstdetection property or parameter (for example, an emission and/orexcitation spectral maximum), which also labeled a secondoligonucleotide probe with a second label having a second detectionproperty or parameter that is different (i.e., discreet or discernablefrom the first label. The use of multiplexing assays, particularly inthe context of genetic amplification/detection protocols are well-knownto those of ordinary skill in the molecular genetic arts.

The term “vector,” as used herein, refers to a nucleic acid molecule(typically comprised of DNA) capable of replication in a host celland/or to which another nucleic acid segment can be operatively linkedso as to bring about replication of the attached segment. A plasmid,cosmid, or a virus is an exemplary vector.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Next Generation rAAV2 Vectors: Point Mutations in TyrosinesLead to High-Efficiency Transduction at Lower Doses

The present example demonstrates that mutations of surface-exposedtyrosine residues on AAV2 capsids circumvents the ubiquitination step,thereby avoiding proteasome-mediated degradation, and resulting inhigh-efficiency transduction by these vectors in human cells in vitroand murine hepatocytes in vivo, leading to the production of therapeuticlevels of human coagulation factor at reduced vector doses. Theincreased transduction efficiency observed for tyrosine-mutant vectorsis due to lack of ubiquitination, and improved intracellular traffickingto the nucleus. In addition to yielding insights into the role oftyrosine phosphorylation of AAV2 capsid in various steps in the lifecycle of AAV2, these studies have resulted in the development of novelAAV2 vectors that are capable of high-efficiency transduction at lowerdoses.

Materials and Methods

Recombinant AAV2 Vectors.

Highly purified stocks of scAAV2 vectors containing the enhanced greenfluorescence protein (EGFP) gene driven by the chicken β-actin (CBA)promoter (scAAV2-EGFP), and ssAAV2 vectors containing the factor IX(F.IX) gene under the control of the apolipoprotein enhancer/human α-1antitrypsin (ApoE/hAAT) promoter (ssAAV2-F.IX) were generated usingpublished methods.

Localization of Surface-Tyrosines on the AAV2 Capsid.

The crystal structure of AAV2 (PDB accession number 1lp3) was used tolocalize the tyrosine residues on the AAV2 capsid surface. Theicosahedral two-, three- and five-fold related VP3 monomers weregenerated by applying icosahedral symmetry operators to a referencemonomer using Program O on a Silicon graphics Octane workstation. Theposition of the tyrosine residues were then visualized and analyzed inthe context of a viral asymmetric unit using the program COOT, andgraphically presented using the program PyMOL Molecular Graphics System(DeLano Scientific, San Carlos, Calif., USA).

Construction of Surface-Exposed Tyrosine Residue Mutant AAV2 CapsidPlasmids.

A two-stage procedure, based on QuikChange II® site-directed mutagenesis(Stratagene, La Jolla, Calif., USA) was performed using plasmid pACG-2.Briefly, in stage one, two PCR extension reactions were performed inseparate tubes for each mutant. One tube contained the forward PCRprimer and the other contained the reverse primer. In stage two, the tworeactions were mixed and a standard PCR mutagenesis assay was carriedout as per the manufacturer's instructions. PCR primers were designed tointroduce changes from tyrosine to phenylalanine residues as well as asilent change to create a new restriction endonuclease site forscreening purposes. All mutants were screened with the appropriaterestriction enzyme and were sequenced prior to use.

Preparation of Whole Cell Lysates (WCL) and Co-Immunoprecipitations.

Approximately 2×10⁶ HeLa cells, mock-treated or treated with MG132, werealso subjected to mock-infection or infection with the WT scAAV2-EGFP orY730F mutant vectors at 5×10³ particles/cell for 2 hr at 37° C. Forimmunoprecipitations, cells were treated with 0.01% trypsin and washedextensively with PBS. WCL were cleared of non-specific binding byincubation with 0.25 mg of normal mouse IgG together with 20 μl ofprotein G-agarose beads. After preclearing, 2 μg of capsid antibodyagainst intact AAV2 particles (mouse monoclonal IgG₃, clone A20;Research Diagnostics, Inc. (Flanders, N.J., USA), or 2 μg of normalmouse IgG (as a negative control) were added and incubated at 4° C. for1 hr, followed by precipitation with protein G-agarose beads. Forimmunoprecipitations, resuspended pellet solutions were used forSDS-PAGE. Membranes were treated with monoclonal HRP-conjugated anti-Ubantibody (1:2,000 dilution) specific for ubiquitin (Ub) (mousemonoclonal immunoglobulin G₁ γIgG₁], clone P4D1; Santa Cruz, Calif.,USA). Immuno-reactive bands were visualized using chemiluminescence(ECL-plus, Amersham Pharmacia Biotech, Piscataway, N.J., USA).

Isolation of Nuclear and Cytoplasmic Fractions from HeLa Cells.

Nuclear and cytoplasmic fractions from HeLa cells were isolated andmock-infected or recombinant wt scAAV2-EGFP or Y700F vector-infectedcells were used to isolate the cytoplasmic and nuclear fractions. Thepurity of each fraction was determined to be >95%.

Southern Blot Analysis for AAV2 Trafficking.

Low-M_(r) DNA samples from nuclear and cytoplasmic fractions wereisolated and electrophoresed on 1% agarose gels or 1% alkaline-agarosegels followed by Southern blot hybridization using a ³²P-labeledEGFP-specific DNA probe.

Recombinant AAV2 Vector Transduction Assays In Vitro.

Approximately 1×10⁵ HeLa cells were used for transductions withrecombinant AAV2 vectors. The transduction efficiency was measured 48-hrpost-transduction by EGFP imaging using fluorescence microscopy. Imagesfrom three to five visual fields were analyzed quantitatively by ImageJanalysis software (NIH, Bethesda, Md., USA). Transgene expression wasassessed as total area of green fluorescence (pixel²) per visual field(mean±SD). Analysis of variance (ANOVA) was used to compare between testresults and the control and they were determined to be statisticallysignificant.

Recombinant AAV2 Vector Transduction Studies In Vivo.

scAAV2-EGFP vectors were injected intravenously via the tail vein intoC57BL/6 mice at 1×10¹⁰ virus particles per animal. Liver sections fromthree hepatic lobes of the mock-injected and injected mice 2 weeks afterinjection were mounted on slides. The transduction efficiency wasmeasured by EGFP imaging as described. ssAAV2-FI.X vectors were injectedintravenously (via the tail vein) or into the portal vein of C57BL/6,BALB/c, and C3H/HeJ mice at 1×10¹⁰ or 1×10¹¹ virus particles per animal.Plasma samples were obtained by retro-orbital bleed and analyzed forhF.IX expression by ELISA.

Results

Mutations in Surface-Exposed Tyrosine Residues Significantly ImproveTransduction Efficiency of AAV2 Vectors.

To demonstrate that tyrosine-phosphorylation of AAV2 capsids leads toincreased ubiquitination and results in impaired intracellulartrafficking, and is therefore unfavorable to viral transduction,surface-exposed tyrosine residues were modified on AAV2 capsids.Inspection of the capsid surface of the AAV2 structure revealed sevensurface-exposed tyrosine residues (Y252, Y272, Y444, Y500, Y700, Y704,and Y730). Site-directed mutagenesis was performed for each of the seventyrosine residues, which were conservatively substituted withphenylalanine residues (tyrosine-phenylalanine, Y-F) (Table 1).scAAV2-EGFP genomes encapsidated in each of the tyrosine-mutant capsidswere successfully packaged, and mutations of the surface-exposedtyrosine residues did not lead to reduced vector stability.

TABLE 1 TITERS OF WILDTYPE (WT) AND TYROSINE- MODIFIED Y→F MUTANT AAV2VECTORS 1^(st) packaging 2^(nd) packaging 3^(rd) packaging 4^(th)packaging AAV Vectors titers (vgs/mL) titers (vgs/mL) titers (vgs/mL)titers (vgs/mL) WT scAAV2-EGFP 3.4 × 10¹¹ 1.0 × 10¹² 3.2 × 10¹¹ 3.0 ×10¹¹ Y252F scAAV2-EGFP 3.8 × 10¹¹ 4.0 × 10¹¹ ND ND Y272 scAAV2-EGFP 7.7× 10¹¹ 1.0 × 10¹¹ ND ND Y444F scAAV2-EGFP 9.7 × 10¹⁰ 4.0 × 10¹⁰ 6.0 ×10⁹  5.0 × 10¹⁰ Y500F scAAV2-EGFP 8.8 × 10¹⁰ 2.0 × 10⁹  4.0 × 10¹⁰ 6.0 ×10¹⁰ Y700F scAAV2-EGFP 1.0 × 10¹¹ 4.0 × 10¹¹ ND ND Y704F scAAV2-EGFP 6.0× 10¹¹ 2.0 × 10¹¹ ND ND Y730F scAAV2-EGFP 1.2 × 10¹¹ 5.0 × 10¹¹ 1.2 ×10¹¹ 4.0 × 10¹¹ ND = Not done.

The transduction efficiency of each of the tyrosine-mutant vectors wasanalyzed and compared with the WT scAAV2-EGFP vector in HeLa cells invitro under identical conditions. From the results, it was evident thatwhereas mock-infected cells showed no green fluorescence, thetransduction efficiency of each of the tyrosine-mutant vectors wassignificantly higher compared with the WT scAAV2-EGFP vector at 2,000viral particles/cell. Specifically, the transduction efficiency ofY444F, Y500F, Y730F vectors was ˜8- to 11-fold higher than the WTvector.

Mutations in Surface-Exposed Tyrosine Residues Dramatically ImproveTransduction

Efficiency of AAV2 Vectors in Murine Hepatocytes in Vivo.

The efficacy of WT and tyrosine-mutant scAAV2-EGFP vectors was alsoevaluated in a mouse model in vivo. The transduction efficiency oftyrosine-mutant vectors was significantly higher, and ranged between4-29-fold, compared with the WT vector. When other tissues, such asheart, lung, kidney, spleen, pancreas, GI tract (jejunum, colon),testis, skeletal muscle, and brain were harvested from mice injectedwith 1×10¹⁰ particles of the tyrosine-mutant vectors and analyzed, noevidence of EGFP gene expression was seen. Thus, mutations in thesurface-exposed tyrosine residues did not appear to alter theliver-tropism following tail vein injection of these vectors in vivo.

Increased Transduction Efficiency of Tyrosine-Mutant Vectors is Due toLack of Uubiquitination, and Improved Intracellular Trafficking to theNucleus.

To further confirm the hypothesis that EGFR-PTK-mediated phosphorylationof capsid proteins at tyrosine residues is a pre-requisite forubiquitination of AAV2 capsids, and that ubiquitinated virions arerecognized and degraded by cytoplasmic proteasome on their way to thenucleus, leading to inefficient nuclear transport, a series ofexperiments were performed as follows.

In the first study, HeLa C12 cells, carrying adenovirus-inducible AAV2rep and cap genes, were mock infected, or infected with WT, Y444F orY730F scAAV2-EGFP vectors. Whereas mock-infected cells showed no greenfluorescence, and ˜15% of cells were transduced with the WT scAAV2-EGFPvectors in the absence of co-infection with adenovirus, the transductionefficiency of Y444F and Y730F scAAV2-EGFP vectors was increased by ˜9and ˜18-fold, respectively, compared with the WT vector. Interestingly,whereas co-infection with adenovirus led to ˜11-fold increase, thetransduction efficiency of Y444F and Y730F scAAV2-EGFP vectors was notfurther enhanced by co-infection with adenovirus. Since adenovirus canimprove AAV2 vector nuclear transport in HeLa cells, these datasuggested that the surface-exposed tyrosine residues play a role inintracellular trafficking of AAV2, and that their removal leads toefficient nuclear transport of AAV2 vectors.

In a second study, HeLa cells, either mock-treated or treated withTyr23, a specific inhibitor of EGFR-PTK, or MG132, a proteasomeinhibitor, both known to increase the transduction efficiency of AAVvectors, were mock-infected or infected with the WT or Y730F scAAV2-EGFPvectors. Whereas mock-infected cells showed no green fluorescence, and˜5% of cells were transduced with the WT scAAV2-EGFP vectors inmock-treated cells, pretreatment with Tyr23 or MG132 led to an ˜9-foldand ˜6-fold increase in the transduction efficiency, respectively.Although the transduction efficiency of Y730F scAAV2-EGFP vectors wasincreased by ˜14-fold compared with the WT vectors, it was not furtherenhanced by pretreatment with either Tyr23 or MG132. These data stronglysuggest that the absence of surface-exposed tyrosine residues, whichprevented phosphorylation of the mutant vectors, likely preventedubiquitination of the capsid proteins, and these vectors could not berecognized on their way to the nucleus and degraded by the proteasome,which led to their efficient nuclear translocation.

In a third study, HeLa cells, either mock-treated or treated with MG132,were mock-infected or infected with the WT, Y730F, or Y444F scAAV2-EGFPvectors. WCL were prepared 4 hrs post-infection and equivalent amountsof proteins were immunoprecipitated first with anti-AAV2 capsid antibody(A20) followed by Western blot analyses with anti-Ub monoclonalantibody. Whereas ubiquitinated AAV2 capsid proteins (Ub-AAV2 Cap) wereundetectable in mock-infected cells, the signal of ubiquitinated AAV2capsid proteins was weaker in untreated cells, and a significantaccumulation of ubiquitinated AAV2 capsid proteins occurred followingtreatment with MG132. Interestingly, infections with Y730F or Y444Fvectors dramatically decreased the extent of accumulation ofMG132-induced ubiquitinated AAV2 capsid proteins. These resultssubstantiate that mutation in tyrosine residues circumventsproteasome-mediated degradation of the vectors.

In a fourth study, the fate of the input WT, Y444F, and Y730F vectorviral DNA was determined in HeLa cells. Southern blot analysis oflow-M_(r) DNA samples isolated from cytoplasmic [C] and nuclear [N]fractions and densitometric scanning of autoradiographs, revealed that˜36% of the input scAAV2 DNA was present in the nuclear fraction incells infected with the WT vector. Interestingly, however, the amount ofinput Y730F and Y444F scAAV2 vector DNA in the nuclear fraction wasincreased to ˜72% and ˜70%, respectively. These results furtherdocumented that mutations in the surface-exposed tyrosine residuesprevent ubiquitination of AAV2 capsids, resulting in a decrease ofproteasome-mediated degradation, and in turn, facilitate nucleartransport of AAV2 vectors.

Tyrosine-Mutant Vectors Express Therapeutic Levels of Human Factor IXProtein at ˜10-Fold Reduced Vector Dose in Mice.

It was important to examine whether tyrosine-mutant AAV2 vectors werecapable of delivering a therapeutic gene efficiently at a reduced vectordose in vivo. To this end, a single-stranded, hepatocyte-specific humanFactor IX (h.FIX) expression cassette was encapsidated in the Y730Fvector, and the efficacy of this vector was tested in three differentstrains of mice (BALB/c, C3H/HeJ, and C57BL/6). Consistently in allthree strains, Y730F vector achieved ˜10-fold higher circulating hF.IXlevels compared with the WT vector following tail vein or portal veinadministration, with the latter being the more effective route. Theseresults clearly indicated that the Y730F vectors expressed therapeuticlevels of human F.IX protein (˜50 ng/mL) at ˜10-fold reduced vector dose(10¹⁰ particles/mouse) in C57BL/6 mice by port vein injection. It shouldbe noted that hepatic viral gene transfer in C57BL/6 mice is generallymore efficient than in the other two strains.

These results demonstrated here are consistent with the interpretationthat EGFR-PTK-induced tyrosine phosphorylation of AAV2 capsid proteinspromotes ubiquitination and degradation of AAV2, thus leading toimpairment of viral nuclear transport and decrease in transductionefficiency. Mutational analyses of each of the seven surface-exposedtyrosine residues yield AAV2 vectors with significantly increasedtransduction efficiency in vitro as well as in vivo. Specifically, Y444Fand Y730F mutant vectors bypass the ubiquitination step, which resultsin a significantly improved intracellular trafficking and delivery ofthe viral genome to the nucleus.

Despite long-term therapeutic expression achieved in preclinical animalmodels by AAV2 vectors composed of the WT capsid proteins, in a recentgene therapy trial, two patients with severe hemophilia B developedvector dose-dependent transaminitis that limited duration ofhepatocyte-derived hF.IX expression to <8 weeks. Subsequent analysesdemonstrated presence of memory CD8⁺ T cells to AAV capsids in humansand an MHC I-restricted, capsid-specific cytotoxic T lymphocyte (CTL)response in one of the hemophilia B patients, which mirrored the timecourse of the transaminitis. It was concluded that this CD8⁺ T cellresponse to input capsid eliminated AAV2-transduced hepatocytes. Thesedata demonstrated that a lower capsid antigen dose is sufficient forefficient gene transfer with the Y730F vector, and show much-reducedubiquitination of AAV-Y730F compared to WT capsid, a prerequisite forMHC I presentation. Thus, the T-cell response to AAV2 capsid (a serioushurdle for therapeutic gene transfer in the liver), may be avoided byusing the surface-exposed tyrosine-mutant AAV2 vectors.

Dramatically increased transduction efficiency of tyrosine-mutantvectors have also been observed in primary human neuronal andhematopoietic stem cells in vitro and in various tissues and organs inmice in vivo. Double, triple, and quadruple tyrosine-mutants have alsobeen constructed to examine whether such multiple mutants are viable,and whether the transduction efficiency of these vectors can beaugmented further. It is noteworthy that with a few exceptions (Y444positioned equivalent to a glycine in AAV4 and arginine in AAV5; Y700positioned equivalent to phenylalanine in AAV4 and AAV5; and Y704positioned equivalent to a phenylalanine in AAV7), these tyrosineresidues are highly conserved in AAV serotypes 1 through 10.

Example 2 Activation of the NF-κB Pathway by rAAV Vectors

Since the in silico analysis with human transcription factor databasedemonstrated the presence of several binding sites for NF-κB, a centralregulator of cellular immune and inflammatory responses, in theadeno-associated virus (AAV) genome, the present example investigateswhether AAV utilizes NF-κB during its life cycle. Small moleculemodulators of NF-κB were used in HeLa cells transduced with recombinantAAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediatedtransgene expression up to 25-fold. Of the two NF-κB inhibitors (Bay11),which blocks both the canonical and the non-canonical NF-κB pathways,totally ablated the transgene expression, whereas pyrrolidonedithiocarbamate (PDTC), which interferes with the classical NF-κBpathway, had no effect. Western blot analyses confirmed the abundance ofthe nuclear p52 protein component of the non-canonical NF-κB pathway inthe presence of VP16, which was ablated by Bay11, suggesting that thenon-canonical NF-κB pathway is triggered during AAV infection. Similarresults were obtained with primary human dendritic cells (DCs) in vitro,in which cytokines-induced expression of DC maturation markers, CD83 andCD86, was also inhibited by Bay11. Administration of Bay11 prior to genetransfer in normal C57BL/6 mice in vivo resulted in up to 7-folddecrease in AAV vector-induced production of pro-inflammatory cytokinesand chemokines such as, IL-1β, IL-6, TNFα, IL-12β, KC, and RANTES. Thesestudies suggested that transient immuno-suppression with NF-κBinhibitors prior to transduction with AAV vectors leads to a dampenedimmune response, which has significant implications in the optimal useof AAV vectors in human gene therapy.

Recent studies have begun to define the initial activation signals thatresult from AAV gene transfer. One study found AAV-induced signalingthrough the Toll-like receptor 9 (TLR9)-myeloid differentiation factor88 (MyD88) pathway to induce a type I interferon response inplasmacytoid dendritic cells (pDCs), thereby driving subsequent adaptiveimmune responses to the vector and transgene product upon gene transferto murine skeletal muscle (Zhu et al., 2009). These data indicatesensing of the DNA genome by the endosomal TLR9 receptor in pDCs. Noevidence for induction of pro-inflammatory cytokines following in vitropulsing of DCs or macrophages with AAV was found. Still, earlier reportsdemonstrated a rapid, albeit highly transient, Kupffer cell-dependentinnate response to AAV vectors in the liver, which included expressionof several inflammatory cytokines (Zaiss and Muruve, 2008; Zaiss et al.,2008; Zaiss and Muruve, 2005; Zaiss et al., 2002).

Interestingly, the role of NF-κB, a key cellular responder to manystress- and pathogen-derived signals and regulator of pro-inflammatorycytokine expression (Hayden and Ghosh, 2004; Hiscott et al., 2006; Liand Verma, 2002), has not been previously studied in the AAV life cycle.In this example, it is shown that infection of human cells with AAV canlead to activation of the non-canonical NF-κB pathway. In addition,activation of NF-κB substantially increases transgene expression(including in DCs), while inhibition of NF-κB blunts expression.Prevention of inflammatory cytokine induction by transient inhibition ofNF-κB reveals a role for NF-κB in the innate response to AAV in vivo,and importantly, does not interfere with long-term transgene expression.

Results

AAV-ITRs Contain Binding Sites for NF-κB-Responsive TranscriptionFactors.

The existence of a cellular protein which interacts specifically withthe single-stranded D[−]-sequence in the left inverted terminal repeat(ITR) of the AAV2 genome has been previously described (Qing et al.,1997). Since the ssD[+]-sequence in the right ITR is complementary tothe ssD[−]-sequence in the left ITR, it was reasoned that a putativecellular protein might also exist, and interact with the ssD[+]-sequencein the right ITR. In electrophoretic mobility-shift assays, using thessD[+]-sequence probe, a distinct cellular protein was indeed detected,which was designated as ssD[+]-sequence binding protein (ssD[+]-BP)(Qing et al., 1997). Following purification and mass spectrometry,ssD[+]-BP was found to have partial amino acid homology to a cellularNF-κB repressing factor, a negative regulator of transcription.Additional in silico analysis with human transcription factor database[TRANSFAC] demonstrated the presence of several binding sites for NF-κBbinding co-factors, such as p300, TFIIB, and SplI. One of these is thep300/CREB transcription factor that has been recently shown to beassociated with the AAV genome (Dean et al., 2009). Although it is notknown whether the NF-κB signaling is activated by AAV binding to thecell surface receptors/co-receptors, recent studies have demonstratedthat the innate immune response could be triggered either a) through theToll like receptor 9 (TLR9)-myeloid differentiation factor 88 (MYD88)pathway, or b) through the activation of the CD40 ligand on the cellsurface in mouse models in vivo (Zhu et al., 2009; Mays et al., 2009).Both of these ligands are known to interact down-stream with NF-κBtranscription factors during their biological activation (Mineva et al.,2007; Loiarro et al., 2005). The following data demonstrated that theNF-κB is involved in the AAV life cycle.

AAV Infection Activates Non-Canonical NF-κB Pathway in Human Cells.

Small molecule activators and inhibitors of NF-κB signaling were used inHeLa cells transduced with a self-complementary serotype 2 vectorexpressing EGFP (scAAV-EGFP). VP16, an NF-κB activator (Wu and Miyamoto,2008), augmented EGFP expression by ˜25-fold (FIG. 1A and FIG. 1B).Between the two inhibitors tested, Bay11, that blocks the activity ofboth IKKκ and IKKκ, totally ablated EGFP expression, whereas PDTC, whichinhibits IKB degradation by blocking IKB ubiquitin ligase in theclassical pathway (Cuzzocrea et al., 2002), had no noticeable effect onEGFP expression (FIG. 1A and FIG. 1B). Furthermore, VP16-mediatedaugmented transgene expression was completely ablated by Bay11, but notby PDTC (FIG. 6A). Similar results were obtained with both ssAAV vectors(FIG. 6B) and with the tyrosine triple-mutant scAAV vector(Y730+500+444F; TM-AAV), which were described in the previous examples(Markusic et al., 2010) (FIG. 6C). It was concluded, therefore, thattransgene expression from the AAV vector was regulated by thealternative (non-canonical) pathway of NF-κB. This conclusion wasconfirmed by Western blot analysis (FIG. 6D, and FIG. 6E), whichrevealed an increase in the cytosolic p100 and the nuclear p52 proteincomponents of the non-canonical NF-κB pathway by ˜3- to 6-fold in thepresence of VP16. Moreover, transduction with AAV vector by itself(i.e., in the absence of activator) increased p100 and p52 (FIG. 1C),indicating that infection of the cell activated the alternative NF-κBpathway. This increase was ablated by Bay11 treatment, while p65, themarker used for the classical NF-κB pathway, was unaffected (FIG. 1C).

NF-κB Pathway is Operational in Primary Human Antigen-Presenting Cells.

Following AAV Infection.

In primary human dendritic cells (DCs), on the other hand, whiletransgene expression was again substantially increased with the NF-κBactivator (FIG. 2A), AAV infection by itself did not activate NF-κB(FIG. 2B). In the presence of VP16, ˜20-fold increase in EGFP expressionwas observed compared with scAAV vector-transduced DCs. Treatment withcytokines (TNF-α, IL-6, IL-1β, PGE2), known to activate the NF-κBpathway, led to a further increase in transgene expression to ˜26%,which was reduced to ˜12% following treatment with Bay11 (FIG. 2A).Western blot analyses of nuclear fractions further corroborated that thealternative pathway of NF-κB activation (accumulation of p52 proteins)was operational (FIG. 2B). Similar results were obtained following scAAVvector-mediated gene delivery to murine livers in vivo (FIG. 7). Theinventors also tested the capability of NF-κB modulators to inducephenotypic changes in DCs. Flow cytometric analyses of two DC maturationmarkers, CD83 and CD86 indicated that VP16 alone was not able to inducematuration or enhance the expression of co-stimulatory molecules whenused together with the cytokines cocktail. However, treatment with Bay11led to inhibition of cytokine-mediated maturation of APCs, furtherimplicating the involvement of NF-κB (Table 2). This reduction ofmaturation markers expression diminishes the main function of DCs toprocess antigenic material and reduces T-cell activation andproliferation. Thus, it was hypothesized that suppression of NF-κBactivation prior to vector administration might lead to a dampenedinnate immune response against AAV.

TABLE 2 FACS ANALYSES OF MARKERS OF MATURATION OF PRIMARY HUMANDENDRITIC CELLS Geometric means of levels of expression in cellsexpressing Group CD83 CD86 Immature DCs 10.38 7.04 DCs - No maturationsupplement 18.08 13.63 Mature DCs + Cytokines 20.60 26.80 DCs + AAV18.29 12.65 DCs + VP16 16.48 13.70 Mature DCs + AAV + Cytokines 24.2523.75 Mature DCs + AAV + Cytokines + 19.92 21.92 VP16 Mature DCs + AAV +Cytokines + 16.88 10.11 Bay11 Data from a representative experiment areshown (n = 3).

Inhibition of NF-κB Activation Leads to Suppression of Pro-InflammatoryCytokine Production Prior to AAV Vector-Mediated Gene Transfer in Micein Vivo. In in vivo studies, a single dose of Bay11 at 20 mg/kg bodyweight was administered intra-peritoneally (i.p.) 12 hrs prior to vectoradministration in C57BL/6 mice. Transcript levels from liver homogenatesof innate immune mediators (FIG. 3A) or for activation of NF-κB (FIG.3B) genes were measured from Bay11- and vector-injected groups andcompared with sham-injected mice. These data revealed that 2 hrspost-vector administration, mice injected with Bay11+AAV vector hadsignificantly reduced levels of pro-inflammatory cytokines or chemokinesincluding IL-1α, IL-6, TNFα, IL-12α, KC, and RANTES, compared with sham-and AAV vector-injected animals (FIG. 3A), and additionally, theup-regulation of the NF-κB gene expression profile was prevented (FIG.3B). A similar down-regulation trend of these innate immune responsemarkers was seen in mice injected with the more efficacious tyrosinetriple-mutant AAV vector (Y730+500+444F; TM-AAV). The up-regulation oftype I interferon expression by both wild-type (WT-AAV) and TM-AAVvectors was unaffected by Bay11 (FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D,FIG. 8E, and FIG. 8F). Administration of Bay11 also significantlyreduced the anti-AAV2 antibody response in these mice (FIG. 9). The sumof these results implies that the transient inflammatory cytokineresponse, typically seen during in vivo hepatic AAV gene transfer, ismediated by NF-κB activation.

AAV Vector-Mediated Transgene Expression in Murine Hepatocytes. In viewof the observation that Bay11 strongly inhibits AAV-mediated transgeneexpression in HeLa cells in vitro 48 hrs post-transduction (FIG. 1A andFIG. 1B), which would be counter-productive to achieve long-termtransgene expression in vivo, it was important to examine the effect ofBay11 in mice. As can be seen in FIG. 4A, animals injected with orwithout Bay11 had similar levels of EGFP expression from either vectorwhen analyzed 2 weeks after gene transfer. Transduction efficiency ofthe TM-AAV vector was ˜12-fold higher than that of the WT-AAV vector(FIG. 4B), consistent with recently published studies (Markusic et al.,2010). These data suggested that Bay11 administration could safely andeffectively down-regulate mediators of innate immune response withoutcompromising long-term transgene expression.

Materials and Methods

Recombinant AAV Vectors.

Highly purified stocks of self-complementary (sc) AAV2 vectors weregenerated containing either the wild-type (WT) plasmid or the tripletyrosine-mutant (TM; Y730+500+444F) plasmid and the enhanced greenfluorescence protein (EGFP) gene driven by the chicken β-actin (CBA)promoter (WT-scAAV2-EGFP, TM-scAAV2-EGFP) by triple transfection ofHEK-293 cells. The vectors were then purified by CsCl gradientcentrifugation, filter sterilized, and quantified by slot-blothybridization as described (Liu et al., 2003; Kube and Srivastava,1997). The tyrosine-mutant pACG2-Y730+500+444F-Rep/Cap plasmid has beendescribed recently (Markusic et al., 2010).

Recombinant AAV Vector Transduction Assays in Vitro.

Optimal concentration of NF-κB-modulating compounds was determined by acell viability assay with tenfold-dilutions from the IC₅₀ or were usedas described previously (Wu and Miyamoto, 2008; Kumar et al., 2008).VP16 or Bay11 (10 or 5 μM, final concentration), and PDTC (50 or 25 μMfinal concentration) were used either alone or in activator/inhibitorcombinations. For transduction experiments, approximately 1×10⁵ HeLacells were either pre-treated with these compounds 24 hrs prior tovector infection. Cells were transduced with 500 or 2,000 vector genomes(vgs) per cell of recombinant WT-AAV or TM-AAV vectors encoding the EGFPtransgene as described previously (Markusic et al., 2010). After 7 daysof culture, primary human dendritic cells were transduced with AAVvectors at 2000 vgs/cell and incubated for 48 hrs. Transgene expressionwas assessed as total area of green fluorescence (pixel) per visualfield (mean±SD), or by flow cytometry. Analysis of variance (ANOVA) wasused to compare between test results and the control and they weredetermined to be statistically significant.

Recombinant AAV Vector Transduction Studies In Vivo.

Groups of 6-weeks old normal C57BL/6J mice (Jackson Laboratories, BarHarbor, Me., USA) were administered intra-peritoneally, with a singledose (20 mg/kg) of NF-κB inhibitor Bay11, in a 200-μL volume diluted inDMSO (day 0). Animals injected with only the DMSO carrier solvent wereconsidered as baseline (mock) group (n=75) and animals injected withBay11 were the test group (n=75). At this point, the animals from mockand Bay11 groups were randomized to receive either phosphate bufferedsaline (PBS, pH 7.4) or WT-AAV or TM-AAV vectors (n=25 mice each group).On day 1, ˜1×10¹¹ viral genome (vg) particles of WT-AAV2-EGFP orTM-AAV2-EGFP vectors or PBS were administered intravenously via the tailvein. To measure the modulation of immune response to AAV, 5 animalseach from PBS-, WT-AAV-, or TM-AAV vector-injected groups weresacrificed by carbon-dioxide inhalation at different time pointspost-vector administration (2, 6, 10, 24 hrs and day 10). Hepatic lobeswere collected, cross-sectioned and mounted on slides to study theeffect of Bay11 on AAV-mediated EGFP expression (from day 10 mice). Allanimal studies were conducted in accordance with institutional animalcare and use committee guidelines.

Gene-Expression Analysis of Innate Immune Response by RT-PCR Assay.

Groups of 6-weeks old normal C57BL/6J mice were administeredintra-peritoneally, with a single dose (20 mg/kg) of NF-κB inhibitor,Bay11, in a 200-μL volume diluted in DMSO (day 0). On day 1, mice wereinjected with either phosphate-buffered saline (PBS, pH 7.4), or with˜1×10¹¹ vgs of the wild-type (WT) AAV-EGFP vectors, or the tyrosinetriple-mutant (TM) AAV-EGFP vectors intravenously via the tail-vein (n=5mice each group). At 2 hr post-vector administration, gene expressionprofiling of the innate immune response was performed that includedToll-like receptors 1-9, MyD88, MIP-1, IL-1α, IL-1β, IL-12α, IL6, KC,TNFα, RANTES, MCP-1, IFNα, IFNβ, and IP-10. Data were captured andanalyzed using an ABI Prism 7500 Sequence Detection System with v 1.1Software (Applied Biosystems). The baseline was determined automaticallyfor the 18S rRNA and for other genes. Thresholds were determinedmanually for all genes. Gene expression was measured by the comparativethreshold cycle (Ct) method. The parameter threshold cycle (Ct) wasdefined as the cycle number at which the reporter fluorescence generatedby the cleavage of the probe passed a fixed threshold above baseline.Cytokine gene expression was normalized using the endogenous reference18S rRNA gene and mock-infected murine mRNA were used as referencesample. Relative gene expression was determined for each group oftreated and untreated animals and values >2.6 and <0.38 were consideredas significant up-regulations and down-regulations between the groupsand was calculated by assessing the variability in the 96-well platesused to measure specific gene expression.

Cells, Antibodies and Chemicals.

HeLa cells were obtained from the American Type Culture Collection(Rockville, Md., USA) and maintained as monolayer cultures inIscove's-modified Dulbecco's medium (IMDM, Invitrogen Carlsbad, Calif.,USA) supplemented with 10% newborn calf serum (NCS) (Lonza, Inc., Basel,Switzerland) and antibiotics. Leukapheresis-derived PBMCs wereresuspended in serum-free AIM-V medium (Lonza) and semi-adherent cellfractions were incubated in serum-free AIM-V medium supplemented withrecombinant human IL-4 (500 U/mL) and GM-CSF (800 U/mL) (R&D Systems,MN, USA). Cells were treated with NF-κB modulators (10 mM VP16 or 10 mMBay11), and cytokines cocktail including 10 ng/mL TNF-α, 10 ng/mL IL-1,10 ng/mL IL-6, 1 mg/mL PGE2 (R&D Systems) for 20 hr. Cells wereharvested, characterized to ensure they met the typical phenotype ofmature DCs (CD83, RPE, murine IgG1, CD86, FITC, murine IgG1;Invitrogen). All primary and secondary antibodies were purchased fromCell Signaling Technology, Inc. (Danvers, Mass., USA) or Santa CruzBiotechnology, Inc (Santa Cruz, Calif., USA). NF-kB activators[Etoposide (VP16), Aphidicolin, Hydroxyurea (HU)] and NF-kB inhibitors[Bay11-7082 (Bay11), Pyrrolidine dithiocarbamate (PDTC)] were purchasedfrom Sigma-Aldrich Co. (St. Louis, Mo., USA). These compounds werere-suspended in either DMSO (Sigma-Aldrich) or in sterile, DNAase-,RNAase-free water (Invitrogen) as per the manufacturer's instructions.

Western Blot Analyses.

Homogenized lysates of the cell pellets from ˜2×10⁶ HeLa cells or DCs,mock or pre-treated with the optimal concentration of NF-κB activatorsor inhibitors were used for sample preparation. Whole cell proteins wereisolated using the RIPA lysis buffer (Sigma-Aldrich) and cytoplasmic andnuclear proteins were extracted using a commercial kit (NE-PERExtraction Reagent Kit, Pierce Biotech, Rockford, Ill., USA) as per themanufacturer's protocol in the presence of a protease inhibitor cocktail(Halt™ Protease Inhibitor Cocktail Kit, Pierce Biotech). The proteinextracts were boiled for 5 min under reducing conditions [SDS-samplebuffer containing 62.5 mM Tris-HCl (pH 6.8 at 25° C.), 2% wt./vol. SDS,10% glycerol, 50 mM DTT, 0.01% (wt./vol.) bromo-phenol blue (CellSignaling Technology, Inc.)] and stored at −86° C. until furtheranalysis. Equal volumes of samples were run on 4-15% SDS-PAGE (Bio-Rad,Hercules, Calif., USA). Gels were transferred onto a 0.2-μmnitrocellulose membrane (Bio-Rad) and typically incubated overnight with1:1000 dilution of primary antibodies [p100/52, p65, inhibitorykinase-IκBκ, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Lamin B(Cell Signaling Technology, Inc.), β-actin (Santa Cruz Biotechnology)].The next day, blots were incubated with 1:2,000-1:5,000 of theappropriate anti-idiotypic HRP labeled IgG secondary antibody (SantaCruz Biotechnology) Immunoblot detection was performed using the ECLplus Western blotting detection kit (Amersham Biosciences, Piscataway,N.J., USA). The intensity of the protein bands was measured with AdobePhotoshop CS3 Software® and normalized to proteins levels from thehousekeeping gene products used as loading controls.

The basis for the present study was the finding that the host cellularNF-κB can bind to the 20-bp D-sequence present in the AAV invertedterminal repeats (ITRs) (Qing et al., 1997), which was identified byelectrophoretic mobility-shift assays followed by mass-spectrometry(FIG. 10A and FIG. 10B). The data presented in this example provide thefirst evidence of the involvement of NF-κB in AAV infection. Using avariety of pharmacological modulators, which have been extensively usedby other investigators (Wu and Miyamoto, 2008; Kumar et al., 2008) tostudy the NF-κB signaling pathway, it was shown that the non-canonicalNF-κB pathway is up-regulated following AAV infection. This issignificant considering that activation of the NF-κB transcriptionalprogram is a fundamental immediate early step of inflammatory and immuneactivation (Li and Verma, 2002), and NF-κB signaling represents a primecandidate for viral susceptibility or interference (Hiscott et al.,2006). Viruses which activate NF-κB have been shown to be susceptible toinnate immune response through an interferon response (Vesicularstomatitis virus, Measles virus) (Hiscott et al., 2003), toll-likereceptor (TLR) dependent (Ebola virus, Respiratory syncytial virus)(Okumura et al., 2010; Lizundia et al., 2008), and TLR-independentsignaling pathway (Cytomegalovirus, Hepatitis C virus) (Castanier etal., 2010; Gourzi et al., 2007). On the other hand, many viruses disruptthe innate immune responses and NF-κB using multifunctional viral decoyproteins that target specific aspects of the NF-κB pathway. Viruses,including human immunodeficiency virus type I (HIV-I), human T-cellleukemia virus type 1 (HTLV-1), Human herpesvirus 8 (HHV8) andEpstein-Barr virus (EBV), have incorporated aspects of NF-κB signalinginto their life cycle and pathogenicity, and thus utilize NF-κBactivation to promote their survival (Hiscott et al., 2006).

In contrast, it stands to reason that the non-canonical pathway of NF-κBis activated following AAV infection both because the non-canonicalNF-κB activation is known to be important for innate and adaptive immuneresponse (Gilmore, 2006), and AAV vectors lack complex structural geneelements necessary to develop any NF-κB-like decoy proteins. Theexacerbated activation of the non-canonical pathway has been associatedto a wide range of inflammatory disorders like rheumatoid arthritis,ulcerative colitis or B cell lymphomas (Dejardin, 2006). Monarch-1, apyrin-containing protein expressed exclusively in cells of myeloidlineage suppresses pro-inflammatory cytokines and chemokines throughinhibition of NF-κB inducing kinase (NIK) necessary to activatenon-canonical NF-κB pathway (Lich et al., 2007). The activation ofnon-canonical pathway of NF-κB activation has been shown to result inmaturation and T-cell priming activity of DCs over-expressing a mutatedIκBκ which blocks activation of the classical pathway (Lind et al.,2008). In alymphoplasia (Aly) mouse deficient in NIK, the cross-primingof CD8+ T cells to exogenous antigens in DCs is affected suggesting theimportance of this pathway in adaptive immunity (Lind et al., 2008).Mice deficient in non-canonical pathway components are also deficient insecondary lymphoid organ development and homeostasis (Guo et al., 2008).It is not known whether AAV-binding activates the NF-κB signaling to acell surface receptor. Recent studies have demonstrated that the innateimmune response to AAV could be triggered through the TLR9-MYD88 pathwayor through activation of the CD40 ligand on cell surface in murinemodels in vivo (Zhu et al., 2009; Mays et al., 2009). It is interestingto note that while both rely on NF-κB signaling down-stream for mountingan innate immune response (Mineva et al., 2007; Loiarro et al., 2005),activation of TNF super family receptors such as CD40L can activate thenon-canonical NF-κB pathway (Qing et al., 2005).

Based on the evidence that the first “danger-signal” or “trigger” toimmune surveillance directed against AAV vectors may be the activationof alternative NF-κB signaling pathway, it was reasoned that transientblocking of NF-κB during AAV vector administration could dampen the hostimmune response. One possible strategy to negate the NF-κB-priming byAAV is to generate targeted mutations against the NF-κB responsivetranscription factor binding sites in the AAV-ITRs. However, given thepleiotropic functions of NF-κB proteins in cellular physiology (Haydenand Ghosh, 2004), it is possible that different NF-κB-responsivecytokine promoter-binding transcription factors might be operational indifferent cell types. Alternatively, a protocol for transientimmuno-suppression by targeting the NF-κB pathway might be universallyapplicable. The selective NF-κB inhibitor, Bay11, can markedly reducemarkers of inflammation and innate immune response to AAV vectors yetdoes not affect its transgene expression in vivo. Bay11 was able todown-regulate the activity of several key regulators namely, IL-1α,IL-6, TNFα, IL-12α, KC and RANTES, suggesting the benefit of using thispharmacologic modulator to selectively down-regulate the inflammatoryand innate immune response against AAV vectors. Interestingly, NIK thatis critical for activation of the non-canonical NF-κB pathway, is alsoknown induce activation of IL-1α, IL-6, IL-12α, TNFα and RANTES inresponse to a variety of viral infections (DiPaolo et al., 2009;Yanagawa and Onoe, 2006; Andreakos et al., 2006; Habib et al., 2001). Inaddition, it is well recognized that NIK is pivotal to the activationand function of the quiescent professional antigen presenting cells, theDCs, whose activity is critical for priming of the antigen specific CD4+helper T cells, leading to immune responses to relevant targets such asthe delivery vector (Andreakos et al., 2006; Habib et al., 2001; Martinet al., 2003; Brown and Lillicrap, 2002). In vitro, NIK increases DCantigen presentation by potently activating NF-κB and consequentlyup-regulating the expression of cytokines (TNFα, IL-6, IL-12, IL-15, andIL-18), chemokines {IL-8, RANTES, macrophage inflammatory protein-1α,monocyte chemo-attractant protein-1, and monocyte chemo-attractantprotein-3}, MHC antigen-presenting molecules (class I and II), andco-stimulatory molecules (CD80 and CD86) (Andreakos et al., 2006). Invivo, NIK enhances immune responses against a vector-encoded antigen andshifts them toward a T helper 1 immune response with increased IgG2alevels, T-cell proliferation, IFN-γ production, and cytotoxic Tlymphocyte responses more potently than complete Freund's adjuvant(Andreakos et al., 2006). Bay11, used in this study, prevents theactivity of IKKα and β, which are the substrates for NIK in thenon-canonical pathway (Pierce et al., 1997). These data indicate thehigh specificity of Bay11 in targeting the non-canonical NF-κB pathwayas well as its ability to prevent the activation of major modulators ofimmune response.

A protocol for transient immuno-suppression by targeting the NF-κBpathway might be universally applicable to limit immuno-toxicities.Indeed, a recent report showed decreased AAV capsid antigen presentationby the use of a proteasomal inhibitor, Bortezomib [Velcade®] (Finn etal., 2010). Bortezomib has a considerable anti-myeloma efficacy (Kubeand Srivastava, 1997), which is likely in large part due to repressionof NF-κB signaling. It may therefore be possible to simultaneously blockMHC I presentation of capsid and inflammatory signals or use moreselective NF-κB-targeted therapies, such as Bay11 in this study, or thenewer IKK inhibitors in order to further enhance the safety andtherapeutic efficacy of AAV vectors.

Example 3 Development of Optimized AAV3 Serotype Vectors

Adeno-associated virus 2 (AAV2), a non-pathogenic human parvovirus,contains a single-stranded DNA genome, and possesses a widetissue-tropism that transcends the species barrier (Muzyczka, 1992).Recombinant AAV2 vectors have gained attention as a promising vectorsystem for the potential gene therapy of a variety of human diseases,and are currently in use in a number of gene therapy clinical trials(Daya and Berns, 2008). More recently, several additional AAV serotypeshave been isolated, and have been shown to transduce specific cell typesefficiently (Muramatsu et al., 1996; Chiorini et al., 1997; Chiorini etal., 1999; Rutledge et al., 1998; Gao G P et al., 2002; Vandenberghe etal., 2004). Whereas various steps in the life cycle of AAV2 arereasonably well understood (Summerford and Samulski 1998; Qing et al.,1999; Summerford et al. 1999; Hansen et al., 2000; Hansen et al., 2001;Sanlioglu et al., 2000; Douar et al., 2001; Zhao et al., 2006; Thomas etal. 2004; Zhong et al. 2004; Ferrari et al., 1996; Fisher et al. 1996;Qing et al., 2004; Zhong et al., 2004; Zhong et al., 2004; Zhong et al.,2008; McCarty et al., 2004; Bainbridge et al., 2008), less is knownabout the other serotypes.

Of the 10 commonly used AAV serotypes, AAV3 has been reported totransduce cells and tissues poorly (Zincarelli et al.; Zincarelli etal., 2008). However, recent studies revealed that AAV3 vectors transduceestablished human hepatoblastoma (HB) and human hepatocellular carcinoma(HCC) cell lines as well as primary human hepatocytes extremelyefficiently (Glushakova et al., 2009). Subsequently, it was documentedthat AAV3 infection was strongly inhibited by hepatocyte growth factor(HGF), HGF receptor (HGFR) specific siRNA, and anti-HGFR antibody, whichsuggested that AAV3 utilizes HGFR as a cellular receptor/co-receptor forviral entry (Ling et al., 2010).

The ubiquitin-proteasome pathway plays a crucial role in intracellulartrafficking of AAV vectors (Douar et al., 2001; Zhong et al., 2007; Duanet al., 2000). Intact AAV2 capsids can be phosphorylated at tyrosineresidues by epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK), and that tyrosine-phosphorylation of AAV capsids negativelyaffects viral intracellular trafficking and transgene expression. Theseobservations led to the suggestion that tyrosine-phosphorylation is asignal for ubiquitination of AAV capsids followed by proteasome-mediateddegradation (Duan et al., 2000; Zhong et al., 2008). This led to thehypothesis that mutations of the surface-exposed tyrosine residues (Y)to phenylalanine (F) might allow the vectors to evade phosphorylation,ubiquitination and proteasome-mediated degradation. Indeed, mutations ofthe surface-exposed tyrosine residues in AAV2 vectors led tohigh-efficiency transduction at lower doses both in HeLa cells in vitroand murine hepatocytes in vivo (Zhong et al., 2008). Therapeutic levelsof expression of human factor IX have been obtained in several differentstrains of mice using the single and multiple tyrosine-mutant AAV2vectors (Zhong et al., 2008; Markusic et al., 2010). Additional studieshave corroborated that similar Y-to-F mutations in AAV serotypes 6, 8and 9 also lead to augmented transgene expression (Petrs-Silva et al.,2009; Qiao et al., 2010; Taylor and Ussher, 2010). Six of sevensurface-exposed tyrosine residues in AAV2 are also conserved in AAV3,but their involvement in AAV3-mediated transduction has not beenevaluated.

This example demonstrates that: (i) AAV3 vector-mediated transduction isdramatically increased in T47D cells, a human breast cancer cell linethat expresses undetectable levels of the endogenous hHGFR (Abella etal., 2005), following stable transfection and over-expression of hHGFR;(ii) the tyrosine kinase activity associated with hHGFR negativelyaffects the transduction efficiency of AAV3 vectors; (iii) the use ofproteasome inhibitors significantly improves AAV3 vector-mediatedtransduction; (iv) site-directed mutagenesis of three surface-exposedtyrosine residues on the AAV3 capsid leads to improved transductionefficiency; (v) a specific combination of two tyrosine-mutations furtherimproves the extent of transgene expression; and (vi) AAV3 vectorsefficiently transduce human HB and HCC tumors in a murine xenograftmodel in vivo, following both intratumoral or systemic administration.These optimized AAV3 vectors provide improved tools for gene therapy,and particularly for the therapy of liver cancer in humans.

Materials and Methods

Cell Lines and Cultures.

Human cervical cancer (HeLa) and hepatocellular carcinoma (Huh7) celllines were purchased from American Type Culture Collection (Manassas,Va., USA), and maintained in complete DMEM medium (Mediatech, Inc.,Manassas, Va., USA) supplemented with 10% heat-inactivated fetal bovineserum (FBS, Sigma-Aldrich, St. Louis, Mo., USA), 1% penicillin andstreptomycin (P/S, Lonza, Walkersville, Md., USA). A newly establishedhuman hepatoblastoma (Hep293TT) cell line (Chen et al., 2009) wasmaintained in complete RPMI medium 1640 (Invitrogen, Camarillo, Calif.,USA) supplemented with 15% heat-inactivated FBS (Sigma-Aldrich), 1%penicillin and streptomycin (P/S, Lonza, Walkersville, Md.). Cells weregrown as adherent cultures in a humidified atmosphere at 37° C. in 5%CO₂ and were sub-cultured after treatment with trypsin-versene mixture(Lonza) for 2-5 min at room temperature, washed and re-suspended incomplete medium. A human breast cancer cell line, T47D, and T47D cellsstably transfected with a hHGFR expression plasmid (T47D+hHGFR), weremaintained in complete DMEM medium (Mediatech, Inc.) with or without 600μg/mL of G418, supplemented with 10% heat-inactivated fetal bovine serum(FBS, Sigma-Aldrich, St. Louis, Mo., USA), 1% penicillin andstreptomycin (Lonza).

Recombinant AAV Plasmids and Vectors.

Recombinant AAV3 packaging plasmid and recombinant AAV2-CBAp-EGFP vectorplasmid were generously provided respectively by Drs. R. Jude Samulskiand Xiao Xiao, University of North Carolina at Chapel Hill, Chapel Hill,N.C. Highly purified stocks of scAAV2 and scAAV3 vectors containing theenhanced green fluorescence protein (EGFP) gene driven by the chickenβ-actin promoter (CBAp) were packaged by the calcium phosphatetriple-plasmid transfection protocol described previously (Wu et al.,2007; Kube and Srivastava, 1997). The physical particle titers ofrecombinant vector stocks were determined by quantitative DNA slot-blotanalyses (Kube and Srivastava, 1997).

Construction of Surface-Exposed Tyrosine Residue Mutant AAV3 CapsidPlasmids.

A two-stage procedure, based on QuikChange II® site-directed mutagenesis(Stratagene) was performed by using plasmid pAAV3 as describedpreviously (Glushakova et al., 2009; Ling et al., 2010). Briefly, instage one, two PCR extension reactions were performed in separate tubesfor each mutant. One tube contained the forward PCR primer and the othercontained the reverse primer (Table 3).

In stage two, the two reactions were mixed and a standard PCRmutagenesis assay was carried out as the manufacturer's instructions.PCR primers were designed to introduce changes from tyrosine tophenylalanine residues and a silent change to create a new restrictionendonuclease site for screening purposes (Table 3). All mutants werescreened with the appropriate restriction enzyme and were sequencedbefore use.

TABLE 3 NUCLEOTIDE SEQUENCES OF PRIMERSUSED FOR SITE-DIRECTED MUTAGENESIS Mutants Primer Sequences (5′ to 3′)Y252F ACCAGAACCTGGGCTCTGCCCACTTTCAACAACCAT            ApaI      Tyr→PheCTCTACAAG (SEQ ID NO: 11) Y272F CAATCAGGAGCTTCGAACGACAACCACTTCTTTGGC           +BstBI       Tyr→Phe TACAGCACC (SEQ ID NO: 12) Y444F CTTATCGATCAGTATCTGTACTTCCTGAACAGAACG    +ClaI          Tyr→PheCAAGGAACA (SEQ ID NO: 13) F501Y GCTAACGACAACAACAACAGTAACTAT CCATGGACA                     Phe→Tyr +NcoI GCGGCCAGCAAA (SEQ ID NO: 14) Y701FTGGAATCCAGAGATTCAGTT CACGTCCAACTACAAC              Tyr→Phe +BmgBIAAGTCTGTT (SEQ ID NO: 15) Y705F GAGATTCAGTACACGTCCAACTTCAACAAGTCTGTT          +AflIII   Tyr→Phe AATGTGGAC (SEQ ID NO: 16) Y731FGTGAACCTCGCCCTATTGGAACCCGGTTTCTCACAC                         Tyr→PheGAAACTTG (SEQ ID NO: 17) The codon triplets are shown in bold; red fontsdenote the mutations from tyrosine to phenylalanine residues, and greenfonts indicate the silent mutations to eliminate/create the restrictionenzyme sites (underlined), which were used to identify the desiredclones.

AAV Vector Transduction Assays.

Huh7 or HeLa cells were seeded in 96-well plates at a concentration of5,000 cells per well in complete DMEM medium. AAV infections wereperformed in serum- and antibiotic-free DMEM medium. Hep293TT cells wereseeded in 96-well plates at a concentration of 10,000 cells per well incomplete RPMI medium. The infections were performed in serum- andantibiotic-free RPMI medium. The expression of EGFP was analyzed bydirect fluorescence imaging 72-hrs' post-transduction.

Western Blot Analyses.

Cells were harvested and disrupted in a radio-immunoprecipitation assay(RIPA) lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1%NP-40, 0.25% sodium deoxycholate and 1 mM EDTA with protease inhibitorcocktail, 1 mM NaF and 1 mM Na₃VO₄). Total protein concentration wasmeasured using a Bradford reagent (Bio-Rad) and equal amounts (50 μg) ofwhole cell lysates were resolved by SDS-PAGE. After electrophoresis,samples were electro-transferred to a nitrocellulose membrane (Bio-Rad),probed with relevant primary antibodies at 4° C. overnight, incubatedwith horseradish peroxidase-conjugated secondary antibodies (JacksonImmunoResearch, West Grove, Pa., USA), and detected with an enhancedchemiluminescence substrate (Amersham). Antibodies against phospho-c-Met(Y1234/1235), total c-Met, phospho-Akt (S473) and phospho-ERK(T202/Y204) were purchased from Cell Signaling, and anti-β-actin (AC-74)antibody was obtained from Sigma-Aldrich.

Recombinant AAV3 Vector Transduction Studies in Mouse Xenograft Models.

Groups of 6-weeks old NSG mice (Jackson Laboratories) were injectedsubcutaneously with 5×10⁶ Hep293TT or Huh7 cells. Four-weekpost-injection, indicated numbers of AAV3 vector genomes (vgs) wereadministered either intratumorally or through tail-vein. Four dayspost-vector administration, tumors were resected, cross-sectioned andevaluated for EGFP expression using a fluorescent microscope. Sectionswere also stained with DAPI to visualize the cell nucleus. All animalstudies were conducted in accordance with approved institutionalguidelines.

Statistical Analysis.

Results are presented as mean±standard deviation (SD). Differencesbetween groups were identified using a grouped-unpaired two-taileddistribution of Student's T test. P values <0.05 were consideredstatistically significant.

Results

Human HGFR is Required for AAV3 Infectivity.

AAV3 utilizes human hepatocyte growth factor receptor (HGFR) as acellular co-receptor (Ling et al., 2010). To unequivocally corroboratethis finding, a human breast cancer cell line, T47D, was used thatexpresses undetectable levels of hHGFR (Abella et al., 2005), as well asT47D cells stably transfected with hHGFR expression plasmids(T47D+hHGFR) (Abella et al., 2005). The expression of hHGFR protein inthe established cell line T47D+hHGFR was confirmed by Western blotanalysis (see FIG. 12C). Equivalent numbers of T47D and T47D+hHGFR cellswere transduced with various multiplicities-of-infection (MOI) ofself-complementary (sc) AAV3-CBAp-EGFP vectors under identicalconditions and transgene expression was determined 72 hrpost-transduction. These results, shown in FIG. 11A, document that thetransduction efficiency of AAV3 vectors is ˜8-13-fold higher in cellsthat express hHGFR than those that do not. AAV3 vector-mediatedtransduction of T47D+hHGFR cells could be completely blocked in thepresence of 5 μg/mL of hHGF (FIG. 11B). Taken together, these dataprovide conclusive evidence that cell surface expression of hHGFR isrequired for successful transduction by AAV3 vectors.

Inhibition of HGFR Protein Tyrosine Kinase Activity EnhancesTransduction Efficiency of AAV3 Vectors.

To examine whether in addition to the extracellular domain, theintracellular domain of HGFR, which contains protein tyrosine kinaseactivity, is also involved in AAV3 infection, a further study wasperformed. Binding of its ligand, HGF, results in dimerization of thereceptor and intermolecular trans-phosphorylation of multiple tyrosineresidues in the intracellular domain (Nguyen et al., 1997). T47D+hHGFRcells were treated for two hrs with increasing concentrations of aspecific HGFR kinase inhibitor, BMS-77760707 (BMS) (Schroeder et al.,2009; Dai and Siemann, 2010). Cells were subsequently infected withscAAV3 vectors at 2,000 vgs/cell. These results are shown in FIG. 12A.It is evident that BMS-777607-treatment led to ˜2-fold increase in AAV3transduction efficiency. Although the p-value is higher when BMS-777607was used at the highest concentration of 10 μM, compared with the lowerconcentration of 1 μM, this change is most likely due to drug toxicity.In previous studies, it was reported that BMS-777607 treatment had nosignificant effect on cell growth at doses ≦1 μM. However, doses of 10μM did result in significant reduction in cell proliferation, whichsuggests that this concentration is toxic to cells (Dai and Siemann,2010). In the next experiment, to rule out any possible non-specificnature of this drug, the parental T47D cells were included as a control.Both cell types were treated with 1 μM BMS-777607 for 2 hr and theninfected with scAAV3 vectors at 10,000 vg/cell. The results, shown inFIG. 12B, indicated that whereas BMS-777607-treatment significantlyenhanced AAV3 infectivity in T47D+hHGFR cells, it had no effect in T47Dcells that lack expression of hHGFR.

To examine whether inhibition of the HGFR kinase led to alterations inthe phosphorylation status of specific cellular proteins involved in thedownstream signaling pathway total and phosphorylation levels of theHGFR protein in both T47D and T47D+hHGFR lysates were determinedfollowing a 2-hr drug-incubation period. Activation of signalingpathways downstream from HGFR kinase, ERK1/2 and Akt, were analyzedusing phosphorylation-specific antibodies. These results, shown in FIG.12C, confirmed that whereas little expression of hHGFR occurs in T47Dcells, the level of expression is significantly higher in T47D+hHGFRcells for both total HGFR and phosphorylated HGFR, which is consistentwith previously published reports (Abella et al., 2005). Treatment ofT47D+hHGFR cells with BMS-777607 completely blocked the phosphorylationof HGFR, but not total HGFR. In addition, BMS-777607-treatment had noeffect on the expression of phosphorylated AKT and ERK1/2. These resultssuggest that the enhancement of AAV3 vector infectivity by theBMS-777607-treatment is due to inhibition of HGFR kinase.

To date, only AAV2 has been reported to use hHGFR as a co-receptor (Yanet al., 2002). The roles of hHGFR and hHGFR kinase inhibitor on otherAAV serotypes are not known. To rule out any non-specific enhancement oftransduction by BMS-777607, other serotypes of AAV, which are notdependent on HGFR, as well as AAV2 vectors, were compared fortransduction efficiency following treatment of cells with BMS-777607.These results, shown in FIG. 13, indicate that whereas AAV2 and AAV3vectors can efficiently transduce T47D+hHGFR cells, other serotypes(AAV4-AAV9) can only transduce these cells at a very low efficiency.This result suggests that hHGFR is not involved in the life cycle ofthese AAV serotypes. Treatment of cells with BMS-777607 significantlyincreased the transduction efficiency of both AAV2 and AAV3 vectors, butnot the other AAV serotypes, which suggested that the effect of theBMS-777607-treatment is AAV serotype-specific.

Proteasome Inhibitors Increase the Transduction Efficiency of AAV3Vectors.

Previous studies have shown that proteasome inhibitors, such as MG132,can significantly enhance the transduction efficiency of AAV2 vectors byfacilitating intracellular trafficking (Zhong et al., 2007; Yan et al.,2002). To evaluate whether MG132 can also improve AAV3 trafficking intarget cells, Huh7, a well-established human hepatocellular carcinomacell line (Nakabayashi et al., 1982), and Hep293TT, a recentlyestablished human hepatoblastoma cell line (Chen et al., 2009), wereeither mock-treated or treated with increasing concentrations of MG132.Following a two-hour treatment, cells were infected with scAAV3-EGFPvectors. HeLa cells, treated with 5 μM MG132 and transduced with scAAV2vectors, were included as a positive control. Transgene expression wasdetermined by fluorescence microscopy 72 hrs post-transduction. Thesedata are shown in FIG. 14A and FIG. 14B. As can be seen, pretreatmentwith MG132 significantly increased the transduction efficiency of scAAV2vectors in HeLa cells, which is consistent with previously results(Zhong et al., 2008). Interestingly, a dose-dependent increase in thetransduction efficiency of scAAV3 vectors in both Huh7 and Hep293TTcells occurred following MG132-treatment, suggesting that AAV3 vectorsalso undergo ubiquitination followed by proteasome-mediated degradation.

Previous studies have also shown that inhibition of EGFR-PTK signalingby Tyrphostin 23 (Tyr23), a specific inhibitor of EGFR-PTK (May et al.,1998), modulates the Ub/proteasome pathway, which in turn, facilitatesintracellular trafficking and transgene expression mediated by AAV2vectors (Zhong et al., 2007). Hep293TT cells were mock-treated ortreated with Tyr23 for 2 hr and transduced with scAAV3 vectors. HeLacells, pretreated with Tyr23 and transduced with scAAV2 vectors, wereincluded as appropriate controls. Transgene expression was determined 72hr post-transduction. These results, shown in FIG. 14C and FIG. 14D,indicate that Tyr23-treatment led to a significant increase in thetransduction efficiency of both scAAV2 and scAAV3 vectors. The increasedtransgene expression was independent of vector entry, since there was nosignificant difference in the amounts of internalized viral DNA in thepresence or absence of either MG132 or Tyr23. These results furthercorroborate the involvement of the host cell Ub/proteasome machinery inthe life cycle of AAV3 vectors as well.

Site-Directed Mutagenesis of Surface-Exposed Tyr Residues SignificantlyImproves Transduction Efficiency of scAAV3 Vectors.

In the preceding examples, the inventors have demonstrated that thereare seven surface-exposed tyrosine residues (Y252, Y272, Y444, Y500,Y700, Y704 and Y730) on AAV2 capsids that are phosphorylated by EGFR-PTKand negatively affect the transduction efficiency of AAV2 vectors (Zhonget al., 2008). Alignment of amino acid sequences from AAV2 and AAV3capsids indicated that six of seven tyrosine residues (Y252, Y272, Y444,Y701, Y705 and Y731) are conserved in AAV3 capsid (Table 4).

TABLE 4 SURFACE-EXPOSED TYR RESIDUES ON AAV CAPSIDS, AND SITE-DIRECTEDMUTAGENESIS TO CONVERT THEM TO PHENYLALANINE RESIDUES AAV2 AAV3 Y252Y252→F Y272 Y272→F Y444 Y444→F Y500 F501 Y700 Y701→F Y704 Y705→F Y730Y731→F The surface-exposed tyrosine (Y) residues on AAV2 and AAV3capsids are shown; arrows denote the site-directed mutations from Y tophenylalanine (F) residues on AAV3 capsids.

One tyrosine residue, Y500 in AAV2, is present as F501 in AAV3. Since ithas been shown that Y to F mutations in several AAV serotypes enhancetransgene expression by circumventing ubiquitination andproteasome-mediated degradation (Zhong et al., 2008; Petrs-Silva et al.,2009; Qiao et al., 2010; Taylor and Ussher et al., 2010), it wasreasoned that mutation of F501 back to a tyrosine residue would reducethe transduction efficiency of AAV3 vectors. This hypothesis was testedby generating a mutant AAV3 vector in which the phenylalanine residuewas substituted with a tyrosine residue (F501Y). The transductionefficiency of the mutant vector was compared with its wild-type (WT)AAV3 counterpart using Huh7 cells under identical conditions. As can beseen in FIG. 15A, the extent of the transgene expression mediated by theF501Y mutant vector was reduced by ˜50% compared with the WT AAV3vector.

To further test the hypothesis that tyrosine-mutations on AAV3 capsidswould lead to decreased EGFR-PTK-mediated phosphorylation followed byreduced ubiquitination and impaired proteasome-mediated degradationresulting in increased transgene expression, all six surface-exposedtyrosine residues on AAV3 capsids were modified and substituted withphenylalanine residues (tyrosine-phenylalanine, Y-F). Each of the singletyrosine-mutant vectors encapsidating scAAV2-CBAp-EGFP genomes could besuccessfully packaged. Vector titers for each of the mutants weredetermined by both quantitative DNA slot blots and qPCR, and nosignificant differences in the packaging efficiency were observed. Thetransduction efficiency of each of the tyrosine-mutant vectors wasanalyzed and compared with the WT scAAV3-CBAp-EGFP vector in both Huh7(FIG. 15B) and Hep293TT (FIG. 15C) cells under identical conditions.From these results, it is evident that, the transduction efficiency ofthree of the tyrosine-mutant vectors (Y701F, Y705F and Y731F) issignificantly higher compared with the WT scAAV3 vector. Specifically,the transduction efficiency of Y731F vector was ˜8-fold higher than theWT vector, followed by Y705F (˜3-fold) and Y701F (˜2-fold) vectors.

Multiple Mutations in Surface-Exposed Tyrosine Residues Further Improvethe Transduction Efficiency of AAV3 Vectors.

In the prior examples involving Y-F mutant AAV2 vectors, it was observedthat specific combinations of the most efficient single-mutations ofsurface-exposed tyrosine residues further augmented the transductionefficiency of AAV2 vectors (Markusic et al., 2010). To examine whether asimilar enhancement could be achieved with AAV3 vectors, the followingdouble- and triple-mutant AAV3 vectors were constructed: Y701+731F,Y705+731F, and Y701+705+731F. Each of these mutant vectors was packagedto similar titers, as determined by both quantitative DNA slot blots andqPCR. The transduction efficiency of these multiple-mutants was comparedwith the WT and the Y731F single-mutant AAV3 vectors in Huh7 cells underidentical conditions. These results are shown in FIG. 16A. As can beseen, whereas the Y731F mutation significantly increased thetransduction efficiency of AAV3 vectors, as observed before, only one ofthe double-mutations (Y705+731F) led to an additional significantincrease in transgene expression. Interestingly, the transductionefficiency of both the double mutant (Y701+731F) and the triple mutant(Y701+705+731F) vectors was reduced to levels similar to the WT AAV3vector. The best-performing single and multiple tyrosine-mutants onhuman liver cancer cells were then evaluated for transduction of T47Dand T74D+hHGFR cells (FIG. 16B). Similar to human liver cancer cells,the tyrosine-mutant rAAV3 vectors led to high-efficiency transduction ofboth cell types, with or without hHGFR expression.

To examine the possibility whether the observed enhanced transductionefficiency of the Y-F mutant vectors was due to the involvement of oneor more additional putative cellular receptor/co-receptor functions, theWT, Y731F, and Y705+731F mutant scAAV3-CBAp-EGFP vectors were used totransduce Huh7 cells in the absence or the presence of 5 μg/ml hHGFunder identical conditions. These results are shown in FIG. 16C. As isevident, the presence of hHGF dramatically inhibited the transductionefficiency and transgene expression of all three AAV3 vectors, which isconsistent with the interpretation that the tyrosine-mutant vectors alsoutilize hHGFR as a cellular receptor/co-receptor for viral entry.

AAV3 Vectors Transduce Human Liver Tumors in Murine Xenograft Models.

To demonstrate AAV3 vectors could also transduce human HB and HCC tumorsin a xenograft mouse model in vivo, ˜5×10⁶ HCC (Huh7) or HB (Hep293TT)cells were injected sub-cutaneously in NOD/Scid gamma (NSG) mice.Four-weeks later, when tumors were clearly visible and palpable in bothgroups of animals, ˜2×10¹⁰ vgs of scAAV3-CBAp-EGFP vectors were injecteddirectly into tumors. Four-days post-vector injections, tumors wereexcised and thin sections were examined under a fluorescence microscope.These results indicated that AAV3 vectors were effective to transduceboth human HCC (FIG. 17A) and HB (FIG. 17B) tumors in vivo. Consistentwith the in vitro data, the transduction efficiency of AAV3 vectors washigher in Hep293TT cell-derived tumors than that in Huh7 cell-derivedtumors.

Optimized Tyrosine-Mutant AAV3 Vectors are Highly Efficient inTransducing Human Liver Tumors in Murine Xenografts.

Next, the best performing double tyrosine-mutant AAV3 vectors werefurther evaluated in vivo for xenograft human liver tumors genetransfer. In the first set of studies, ˜5×10¹⁰ vgs of either thewild-type (WT) scAAV3- or Y705+731F-AAV3-CBAp-EGFP vectors wereintratumorally injected in NSG mice bearing human HB (Hep293TT) tumors.Four-days post-vector injections, tumors were excised, and thin sectionswere examined under a fluorescence microscope (FIG. 17C). As can beseen, tumors injected with the WT-AAV3 vectors exhibited detectablelevels expression of EGFP. The transduction efficiency of the doubletyrosine-mutant AAV3 vectors was significantly higher compared with theWT AAV3 vectors, which is consistent with the in vitro data.

In the second set of studies, ˜5×10¹¹ vgs of either theWT-scAAV3-CBAp-EGFP vector or the Y705+731F-scAAV3-CBAp-EGFP vector wereinjected via the tail-vein in NSG mice bearing human HB (Hep293TT)tumors. Phosphate-buffered saline (PBS) injections were used as anappropriate control. Whereas little transgene expression occurred intumors from mice injected with pBS (FIG. 18A), direct tumor-targetingcould be achieved following systemic administration of AAV3 vectors. Thetransduction efficiency of the optimized tyrosine-mutant AAV3 vectors(FIG. 18C), once again, was significantly higher than that of the WTAAV3 vectors (FIG. 18B). These data suggest that the observed increasedtransduction efficiency of tyrosine-mutant AAV3 vectors was independentof viral administration route.

HGFR is a trans-membrane receptor tyrosine kinase, and binding of itsligand, HGF, results in dimerization of the receptor and intermoleculartrans-phosphorylation of multiple tyrosine residues in the intracellulardomain. (Liu et al., 2008) Whereas it is clear that AAV3 capsidinteracts with the extracellular domain of hHGFR, it is less clear,whether AAV3-binding to hHGFR also triggers its activation andphosphorylation of the downstream target proteins. The data does indeeddemonstrate that suppression of the hHGFR-PTK activity leads to a modestincrease in AAV3 vector-mediated transgene expression. In this context,it is of interest to note that the transduction efficiency of AAV3vectors is significantly higher in a more recently established humanhepatoblastoma (HB) cell line, Hep293TT, compared with that in a HB cellline, Huh6, which was established nearly three decades ago. Althoughsubtle differences might exist between the two cell lines, specificmutations have been identified in the tyrosine kinase domain of hHGFR inHep293TT cells, which render it inactive, and that the hHGFR-specifickinase inhibitor, BMS-777607, which augments the transduction efficiencyin Huh6 cells, has little effect on AAV3 transduction efficiency inHep293TT cells.

Despite the utilization of two distinct cellular growth factor receptorsas co-receptors by AAV2 (hFGFR1) and AAV3 (hHGFR), the two serotypesappear to share certain post-receptor entry and intracellulartrafficking pathways. For example, both capsids become phosphorylated attyrosine residues by EGFR-PTK, presumably in the late endosomes,followed by ubiquitination, which leads to proteasome-mediateddegradation. (Zhong et al., 2008) However, although 6 of 7surface-exposed tyrosines in AAV2 are conserved in AAV3, the patterns ofbehavior of the corresponding Y-F mutants are somewhat divergent. Forexample, Y730F (for AAV2) and Y731F (for AAV3) are the most efficientsingle-mutants, followed by Y444F (for AAV2), and Y705F (for AAV3), thetransduction efficiency of Y444F (for AAV3) remains unaltered.Similarly, whereas the transduction efficiency of the Y730+444Fdouble-mutant (for AAV2) is not significantly different from that ofY730F, the transduction efficiency of the Y705+731F double-mutant (forAAV3) is significantly higher than Y731F. Furthermore, the Y730+500+444Ftriple-mutant (for AAV2) is the most efficient, the Y731+501+705Ftriple-mutant (for AAV3) is the most efficient, the Y501 residue havingalready been mutated in the WT AAV3 capsid. Interestingly, even the WTAAV3 vectors were able to transduce human liver tumors reasonably wellin a mouse xenograft model in vivo following intratumor injection.However, evidence that the tyrosine-mutant vector resulted in highergene transfer efficiency in vivo has been demonstrated.

Human liver cancer, especially hepatocellular carcinoma (HCC), is one ofthe most aggressive malignant tumors. The major obstacle to survivalwith HCC is recurrence after HCC resection (Tang, 2005). Thus,transduction of 100% of target cells is desirable in order to completelyeliminate the tumor. In previous studies, it was observed that melittin,a toxic peptide derived from bee venom, inhibits the viability andmotility of HCC cells both in vitro and in vivo via the suppression ofRac1-dependent pathway (Liu et al., 2008) and up-regulation ofmitochondria membrane protein 7A6 (Zhang et al., 2007). Melittin hasbeen shown to induce apoptosis of HCC cells potentially by activatingCaMKII/TAK1/JNK/p38 signaling pathway (Wang et al., 2009).

Based on previous studies with recombinant adenovirus vectors containingthe melittin gene driven by a liver cancer cell-specific promoter toachieve specific killing of liver cancer cells both in vitro and in vivo(Ling et al., 2005), this example provides optimized tyrosine-mutantAAV3-melittin vectors under the control of a liver cancer cell-specificpromoter that can be used to selectively target both primary andmetastatic liver cancer.

Example 4 High-Efficiency Transduction of Human Monocyte-DerivedDendritic Cells by Capsid-Modified Recombinant AAV2 Vectors

Dendritic cells (DCs) are antigen-presenting cells (APCs), which play acritical role in the regulation of the adaptive immune response. DCs areunique APCs and have been referred to as “professional” APCs, since theprincipal function of DCs is to present antigens, and because only DCshave the ability to induce a primary immune response in resting naïve Tlymphocytes. (Banchereau and Steinman, 1998) Although a naturallyoccurring anti-tumor immune response is detectable in patients, thisresponse fails to control tumor growth. On the other hand,monocyte-derived DCs (moDCs) generated ex vivo in the presence ofgranulocyte-macrophage colony-stimulating factor (GM-CSF) andinterleukin 4 (IL-4) possess the capacity to stimulate antigen-specificT-cells after endogenous expression of antigens. (Chapuis et al., 1997;den Brok et al., 2005) For this reason, genetically-modified DCs havebeen extensively studied and numerous Phase I and II clinical trialsevaluating the efficacy of DCs in patients with cancer have beeninitiated. (Figdor et al., 2004; Palucka et al., 2011) However, currentmethods for DC loading are inadequate in terms of cell viability,uncertainty regarding the longevity of antigen presentation, and therestriction by the patient's haplotype. (Palucka et al., 2011)

The possibility of manipulating viral genomes by biotechnologicaltechniques, together with the recent identification of manytumor-associated antigens (TAAs), has sparked an interest in usingrecombinant viruses to express TAAs in the hope of inducing a protectiveantitumor immune response in patients. (Liu, 2010; Robert-Guroff, 2007)Among different methods for gene delivery, vectors based on a humanparvovirus, the adeno-associated virus serotype 2 (AAV2), have attractedmuch attention mainly because of the non-pathogenic nature of thisvirus, and its ability to mediate long-term, sustained therapeutic geneexpression. (Daya and Berns, 2008; Mueller and Flotte, 2008; Srivastava,2008) Successful transduction of different subsets of DCs by differentcommonly used serotypes of AAV vectors has been demonstrated and thepotential advantage of an AAV-based antitumor vaccine discussed.(Pannazhagan et al., 2001; Veron et al., 2007; Mahadevan et al., 2007;Shin et al., 2008; Taylor and Ussher, 2010) However, furtherimprovements in gene transfer by recombinant AAV vectors to DCs in termsof specificity and transduction efficiency are warranted to achieve asignificant impact when used as an anti-tumor vaccine.

Cellular epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK) negatively impacts nuclear transport and subsequent transgeneexpression by recombinant AAV2 vectors primarily due to phosphorylationof capsids at surface tyrosine residues. (Zhong et al., 2007) Thesestudies resulted in the development of next generation recombinant AAV2vectors containing point mutations in surface exposed tyrosine residuesthat transduce various cells and tissues with high-efficiency at lowerdoses compared to the wild-type (WT) vector. (Zhong et al., 2008)However, such single or multiple tyrosine-mutant AAV vectors failed toincrease the transduction efficiency of monocyte-derived DCs (moDCs)more than 2-fold, most likely due to lower levels of expression and/oractivity of EGFR-PTK compared with that in HeLa cells or hepatocytes.(Taylor and Ussher, 2010)

Serine/threonine protein kinases are involved in a wide variety ofcellular processes such as differentiation, transcription regulation,and development of many cell types including immune cells. Such kinasescan also negatively regulate the efficiency of recombinant AAVvector-mediated gene transfer by phosphorylating the surface-exposedserine and/or threonine residues on the viral capsid and target thevectors for proteasome-mediated degradation. In the present example, thefollowing were documented: (i) Site-directed mutagenesis of the 15surface-exposed serine (S) residues on the AAV2 capsid to valine (V)residues leads to improved transduction efficiency of S458V, S492V, andS662V mutant vectors compared with the WT AAV2 vector; (ii) The S662Vmutant vector efficiently transduces human monocyte-derived dendriticcells (moDCs), a cell type not readily amenable to transduction by theconventional AAV vectors; (iii) High-efficiency transduction of moDCs byS662V mutant does not induce any phenotypic changes in these cells; and(iv) Recombinant S662V-vectors encoding a truncated human telomerase(hTERT) gene, used to transduced DCs result in rapid, specific T-cellclone proliferation and generation of robust CTLs, which leads tospecific cell lysis of K562 cells.

Materials and Methods

Cells and Antibodies.

HEK293, HeLa and NIH3T3 cells were obtained from the American TypeCulture Collection and maintained as monolayer cultures in DMEM(Invitrogen) supplemented with 10% FBS (Sigma) and antibiotics (Lonza).Leukapheresis-derived peripheral blood mononuclear cells (PBMCs)(AllCells) were purified on Ficoll-Paque (GEHeathCare), resuspended inserum-free AIM-V medium (Lonza), and semi-adherent cell fractions wereincubated for 7 days with recombinant human IL-4 (500 U/mL) and GM-CSF(800 U/mL) (R&D Systems). Cell maturation was initiated with a cytokinemixture including 10 ng/mL TNF-α, 10 ng/mL IL-1, 10 ng/mL IL-6, and 1mg/mL PGE2 (R&D Systems) for 48 hrs. Prior to EGFP expression cells werecharacterized for co-stimulatory molecules expression to ensure thatthey met the typical phenotype of mature dendritic cells (mDC) (CD80,RPE, murine IgG1; CD83, RPE, murine IgG1; CD86, FITC, murine IgG1;Invitrogen). (Jayandharan et al., 2011)

Site-Directed Mutagenesis.

A two-stage PCR was performed with plasmid pACG2 as described previously(Wang and Malcolm, 1999) using Turbo Pfu Polymerase (Stratagene).Briefly, in stage one, two PCR extension reactions were performed inseparate tubes for the forward and reverse PCR primer for 3 cycles. Instage two, the two reactions were mixed and a PCR reaction was performedfor an additional 15 cycles, followed by DpnI digestion for 1 hr.Primers were designed to introduce changes from serine (TCA or AGC) tovaline (GTA or GTC) for each of the residues mutated.

Production of Recombinant AAV Vectors.

Recombinant AAV2 vectors containing the EGFP gene driven by the chickenβ-actin promoter were generated as described previously (Zologukhin etal., 2002). Briefly, HEK293 cells were transfected usingpolyethelenimine (PEI, linear, MW 25,000, Polyscinces, Inc.).Seventy-two hrs post transfection, cells were harvested and vectors werepurified by iodixanol (Sigma) gradient centrifugation and ion exchangecolumn chromatography (HiTrap Sp Hp 5 mL, GE Healthcare). Virus was thenconcentrated and the buffer exchanged in three cycles to lactatedRinger's using centrifugal spin concentrators (Apollo, 150-kDa cut-off,20-mL capacity, CLP) (Cheng et al., 2011). Ten μL of purified virus wastreated with DNAse I (Invitrogen) for 2 hr at 37° C., then an additional2 hr with proteinase K (Invitrogen) at 56° C. The reaction mixture waspurified by phenol/chloroform, followed by chloroform treatment.Packaged DNA was precipitated with ethanol in the presence of 20 μgglycogen (Invitrogen). DNAse I-resistant AAV particle titers weredetermined by RT-PCR with the following primer-pair, specific for theCBA promoter:

Forward (SEQ ID NO: 18) 5′-TCCCATAGTAACGCCAATAGG-3′, Reverse(SEQ ID NO: 19) 5′-CTTGGCATATGATACACTTGATG-3′

and SYBR Green PCR Master Mix (Invitrogen) (Aslanidi et al., 2009).

Recombinant AAV Vector Transduction Assays In Vitro.

HEK293 or monocyte-derived dendritic cells (moDCs), were transduced withAAV2 vectors with 1,000 vgs/cell or 2,000 vgs/cell respectively, andincubated for 48 hrs. Alternatively, cells were pretreated with 50 μM ofselective serine/threonine kinase inhibitors2-(2-hydroxyethylamino)-6-aminohexylcarbamic acid tert-butylester-9-isopropylpurine (for CaMK-II), anthra[1,9-cd]pyrazol-6(2H)-one,1,9-pyrazoloanthrone (for JNK), and4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole(for MAPK) (CK59, JNK inhibitor 2, PD 98059, Calbiochem), 1 hr beforetransduction. Transgene expression was assessed as the total area ofgreen fluorescence (pixel2) per visual field (mean±SD) as describedpreviously (Markusic et al., 2011; Jayandharan et al., 2011). Analysisof variance was used to compare test results and the control, which weredetermined to be statistically significant.

Western Blot Analysis.

Western blot analysis was performed as described previously. (Akache etal., 2006) Cells were harvested by centrifugation, washed with PBS, andresuspended in lysis buffer containing 50 mM TrisHCl, pH 7.5, 120 mMNaCl, 1% Nonidet P-40, 10% glycerol, 10 mM Na4P2O7, 1 mMphenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, and 1 mM EGTAsupplemented with protease and phosphotase inhibitors mixture (Set 2 and3, Calbiochem). The suspension was incubated on ice for 1 hr andclarified by centrifugation for 30 min at 14,000 rpm at 4° C. Followingnormalization for protein concentration, samples were separated using12% polyacrylamide/SDS electrophoresis, transferred to a nitrocellulosemembrane, and probed with primary antibodies, anti p-p38 MAPK(Thr180/Tyr182) rabbit mAb, total p38 MAPK rabbit mAb and GAPDH rabbitmAb (1:1000, CellSignaling), followed by secondary horseradishperoxidase-linked linked antibodies (1:1000, CellSignaling).

Specific Cytotoxic T-Lymphocytes Generation and Cytotoxicity Assay.

Monocyte-derived dendritic cells (moDCs) were generated as describedabove. Immature DCs were infected with AAV2-S662V vectors encoding humantelomerase cDNA, separated into two overlapping ORF—hTERT838-2229 andhTERT2042-3454 at MOI 2,000 vgs/cell of each. Cells were then allowed toundergo stimulation with supplements to induce maturation. After 48 hr,the mature DCs expressing hTERT were harvested and mixed with the PBMCsat a ratio of 20:1. CTLs were cultured in AIM-V medium containingrecombinant human IL-15 (20 IU/mL) and IL-7 (20 ng/mL) at 20×10⁶ cellsin 25 cm² flasks. Fresh cytokines were added every 2 days. After 7 dayspost-priming, the cells were harvested and used for killing assays(Heiser et al., 2002). A killing curve was generated and specific celllysis was determined by FACS analysis of live/dead cell ratios asdescribed previously (Mattis et al., 1997). Human immortalizedmyelogenous leukemia cell line, K562, was used as a target.

Statistical Analysis.

Results are presented as mean±S.D. Differences between groups wereidentified using a grouped-unpaired two-tailed distribution of Student'sT-test. P-values <0.05 were considered statistically significant.

Results

Inhibition of Specific Cellular Serine/Threonine Kinase IncreasesTransduction Efficiency of rAAV2 Vectors.

In previous studies, inhibition of cellular epidermal growth factorreceptor protein tyrosine kinase (EGFR-PTK) activity and site-directedmutagenesis of the 7 surface-exposed tyrosine residues was shown tosignificantly increase to the transduction efficiency of AAV2 vectors bypreventing phosphorylation of these residues, thereby circumventingubiquitination and subsequent proteasome-mediated degradation of thevectors (Zhong et al., 2008). However, AAV2 capsids also contain 15surface-exposed serine residues, which can potentially be phosphorylatedby cellular serine/threonine kinases widely expressed in various celltypes and tissues. To test the hypothesis that inhibition of such kinaseactivity can prevent phosphorylation of surface-exposed serine residuesand thus improve intracellular trafficking and nuclear transport of AAV2vectors, several commercially available specific inhibitors of cellularserine/threonine kinases were used, including calmodulin-dependentprotein kinase II (CamK-II), c-Jun N-terminal kinase (JNK); andmitogen-activated protein kinase (p38 MAPK). HEK293 cells werepre-treated with specific inhibitors, such as2-(2-hydroxyethylamino)-6-aminohexylcarbamic acid tert-butylester-9-isopropylpurine (for CaMK-II), anthra[1,9-cd]pyrazol-6(2H)-one,1,9-pyrazoloanthrone (for JNK), and4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole(for p38 MAPK) for 1 hr at various concentrations. Cells weresubsequently transduced with either single-stranded (ss) orself-complementary (sc) AAV2 vectors at 1,000 vector genomes (vgs) percell. These results indicated that all inhibitors at an optimalconcentration of 50 μM significantly increased the transductionefficiency of ssAAV2 and scAAV2 vectors, the p38 MAPK inhibitor beingthe most effective (FIG. 19A and FIG. 19B). This observation suggests,albeit does not prove, that the increase in the transduction efficiencywas most likely due to prevention of phosphorylation of vector capsidsrather than improved viral second-strand DNA synthesis.

Site-Directed Mutagenesis of Surface-Exposed Serine Residues on AAV2Capsid Improves AAV2 Vector-Mediated Transgene Expression. The AAV2capsid contains 50 serine (S) residues in the viral protein 3 (VP3)common region of the three capsid VPs, of which 15 (S261, S264, S267,S276, S384, S458, S468, S492, S498, S578, S658, S662, S668, S707, S721)are surface-exposed. (Xie et al., 2002) Each of the 15 S residues wassubstituted with valine (V) by site-directed mutagenesis as described(Zhong et al., 2008). Most mutants could be generated at titers similarto the WT AAV2 vectors, with the exception of S261V, S276V, and S658V,which were produced at ˜10 times lower titers, and S267V and S668V,which produced no detectable levels of DNAse I-resistant vectorparticles. The titers of S468V and S384V mutants were ˜3-5 times higherthan the WT AAV2 vectors. Each of the S-V mutant vectors was evaluatedfor transduction efficiency in HEK293 cells. These results, shown inFIG. 20, indicate that of the 15 mutants, the S662V mutant transducedHEK293 cells ˜20-fold more efficiently than its WT counterpart did. Thetransduction efficiency of the S458V and the S492V mutant vectors wasincreased by ˜4- and 2-fold, respectively. The positions of these threecritical surface exposed serine residues on the AAV2 capsid are shown inFIG. 21A and FIG. 21B. No further increase in transduction efficiencywas observed with the double-mutants (S458+662V and S492+662V), or thetriple-mutant (S458+492+662V), indicating that unlike some of thetyrosine-mutants, combining multiple mutations in the serine residueswas neither additive nor synergistic. Interestingly, the transductionefficiency of the S468V and the S384V mutants, which were produced attiters higher than the WT AAV2 vectors, remained unchanged (S468V) orwere reduced ˜10-fold (S384V) at the same multiplicity of infection(MOI). These data are summarized in FIG. 34.

Substitution of S662 with Different Amino Acids has Diverse Effects onAAV2 Capsid Assembly and AAV2 Vector-Mediated Transgene Expression. Inaddition to S-to-V substitution at position 662, the following 7 mutantswith different amino acids were also generated: S662→Alanine (A),S662→Asparagine (N), S662→Aspartic acid (D), S662→Histidine (H),S662→Isoleucine (I), S662→Leucine (L), and S662→Phenylalanine (F), andevaluated their transduction efficiency in 293 cells. These results,shown in FIG. 22 and summarized in FIG. 35, demonstrate that thesubstitution of S with V led to the production of the most efficientmutant without any change in vector titers. Replacement of S with N, I,L, or F decreased the packaging efficiency ˜10-fold with no significanteffect on the transduction efficiency, whereas substitution with D or Hincreased the transduction efficiency ˜8-fold and ˜4-fold, respectively,with no effect on vector titers. Interestingly, substitution of S to Aincreased the viral titer up to ˜5-fold, and enhanced the transgeneexpression ˜3-fold compared with the WT AAV2 vector. The observedvariability in titers and infectivity of the serine-mutants at position662 suggests the critical role each of the amino acids plays inmodulating both AAV2 packaging efficiency and biological activity.

Transduction Efficiency of S662V Vectors Correlate with p38 MAPKActivity.

Since all of the S662V vector-mediated transgene expression data thusfar were derived using 293 cells, these studies were extended to includethe following cells types: (i) NIH3T3 (mouse embryonic fibroblasts),(ii) H2.35 (mouse fetal hepatocytes), (iii) HeLa (human cervical cancercells), and (iv) primary human monocyte-derived dendritic cells (moDCs).These cell types were transduced with WT scAAV2-EGFP or S662VscAAV2-EGFP vectors at an MOI of 2,000 vgs per cell under identicalconditions. EGFP gene expression was evaluated 48 hrs post-infection(p.i.) for HeLa, 293 and moDCs, and 5 days p.i. for H2.35 and NIH3T3cells. These results are shown in FIG. 23A. As can be seen, although theabsolute differences in the transduction efficiency between WT and S662Vmutant vectors ranged from ˜3-fold (in H2.35 cells) to ˜20-fold (in 293cells) the mutant vector was consistently more efficient in each celltype tested. Since pre-treatment of cells with an inhibitor of cellularp38 MAPK was the most effective in increasing the transductionefficiency (FIG. 19A and FIG. 19B), the inventors examined whether ornot the observed differences in the transduction efficiency of the WTand the mutant vectors was due to variations in the levels of expressionand/or activity of the cellular p38 MAPK. Cell lysates prepared fromeach cell type were analyzed on Western blots probed with specificantibodies to detect both total p38 MAPK and phospho-p38 MAPK levels.GAPDH was used as a loading control. These results, shown in FIG. 23B,indicate that whereas the p38 MAPK protein levels were similar, thekinase activity, as determined by the level of phosphorylation, variedsignificantly among different cell types, and the transductionefficiency of the S662V mutant vector correlated roughly with the p38MAPK activity. These approximate correlations between p38 MAPK activityand the efficiency of the S662V mutant vector can probably be explainedby different cell susceptibilities for AAV infection, the overall numberof viral particles entered cell after primary infection. It remainsunclear as to which precise steps in the life cycle of AAV are modulatedby p38 MAPK-mediated phosphorylation. It is also possible that otherserine/threonine kinases contributing to the difference in efficiency oftransduction by S662V and WT vectors. Interestingly, however,transduction by the WT-AAV2 vectors did not lead to up regulation ofphosphorylation of p38 MAPK in 293 cells or in moDC, further supportinga previous report that AAV does not induce robust phenotypic changes inmoDCs (Markusic et al., 2011).

S662V Vector-Mediated Transduction of Primary Human moDCs does not Leadto Phenotypic Alterations.

MAPK family members play important roles in the development andmaturation of APCs. moDCs, isolated from healthy donor leukapheresis,were treated with 50 μM selective kinase inhibitors as described aboveand then transduced with WT scAAV2-EGFP vectors. Two hrs p.i., cellswere treated with supplements (TNF-α, IL-1β, Il-6, PGE2) to inducematuration. EGFP transgene expression was evaluated 48 hrs p.i. byfluorescence microscopy. Pre-treatment of moDCs with specific inhibitorsof JNK and p38 MAPK increased EGFP expression levels ˜2-fold and˜3-fold, respectively, and the transduction efficiency was enhanced by˜5-fold with the S662V mutant vectors (FIG. 24). Since inhibition ofthese kinases has previously been reported to prevent maturation ofdendritic cells (Beisleve et al., 2005; Nakahara et al., 2006; Nakaharaet al., 2004; Harley, 2008), the capability of S662V mutant to inducephenotypic changes in DCs also was evaluated. moDC were infected with anincreasingly higher MOI up to 50,000 vgs per cell, harvested at 48 hrsp.i., and analyzed by fluorescence-activated cell sorting (FACS) for upregulation of surface co-stimulatory molecules. Flow cytometric analysesof DC maturation markers such as CD80, CD83 and CD86 indicated that,similar to WT AAV2 vectors, the S662V mutant vectors also did not inducethe maturation of moDCs (FIG. 24C). This observation supports thepreviously described low immunogenicity of AAV vectors. (Shin et al.,2008; Jayandharan et al., 2011)

hTERT-Specific CTL Generation by moDC Transduced with AAV2-S662VVectors.

Since the serine-mutant AAV2 vector-mediated transgene expression inmoDC was significantly improved compared with the WT-AAV2 vectors, theability of S662V-loaded moDCs to stimulate the generation of cytotoxicT-lymphocytes and effect specific killing of the target cell wasexamined. Given that human telomerase is recognized as a uniqueanti-cancer target (Harley, 2008; Beatty and Vonderheide, 2008) commonlyexpressed in most cancer cells, a truncated human telomerase (hTERT)gene was cloned under the control of the chicken β-actin promoter andpackaged the DNA into the AAV2 S662V mutant. Non-adherent peripheralblood mononuclear cells (PBMC) containing up to 25% of CD8 positivecells were stimulated once with moDC/hTERT delivered by the S662Vvector. An immortalized myelogenous leukemia cell line, K562, was usedfor a two-color fluorescence assay of cell-mediated cytotoxicity togenerate a killing curve with subsequently reduced effector to targetcell ratio. Result of these experiments, shown in FIG. 25, suggest thatmoDC loaded with hTERT can effectively stimulate specific T cell cloneproliferation and killing activity compared with moDC expressing GFP.Thus, since immunization strategies that generate rapid and potenteffector responses are essential for effective immunotherapy, theseresults support the efficacy of AAV-based delivery methods forvaccination studies.

Discussion

Although the possibility of genetically-modified dendritic cellsstimulating a specific anti-tumor cytotoxic T cell response has beenproven in a number of clinical trials, a reliable method for therapeuticantigen loading, control of expression, and antigen presentation has notyet been previously developed (O'Neill and Bhardwaj, 2007; Tacken etal., 2007). Since the first attempts to transduce dendritic cells withconventional ssAAV vectors nearly a decade ago (Pannazhagan et al.,2001), significant progress has been made in increasing the transductionefficiency of these vectors. For example, the development ofself-complementary AAV (ssAAV) vectors has circumvented a majorrate-limiting step of viral second-strand DNA synthesis, whichdramatically increases transgene expression levels in different subsetsof dendritic cells. (Shin et al., 2008; Aldrich et al., 2006; Wang etal., 2003) AAV vector-based antigen delivery to dendritic cells hassuccessfully been utilized for several cancer models. (Mahadevan et al.,2007; Eisold et al., 2007; Yu et al., 2008)

The natural flexibility of AAV structural and regulatory viralcomponents promotes rapid molecular evolution and formation of numerousserologically distinct serotypes (Gao et al., 2003; Vandenberghe et al.,2009; Wu et al., 2006). Several studies have shown that one can takeadvantage of such plasticity of AAV to generate new vectors withdifferent cell and tissue tropism (Wu et al., 2000; Girod et al., 1999).Other studies revealed that substitution of a single amino acid on theviral capsid can strongly affect viral titer, interaction with cellularreceptor, tissue-tropism and trafficking from endosome to the nucleolus(Zhong et al., 2008; Wu et al., 2006). Wu et al. (2006) have reportedthat replacement of lysine to glutamine at position 531 (K531E) on AAV6capsid reduces gene transfer to mouse hepatocytes in vivo and affinityfor heparin. The reverse mutation (E531K) on AAV1 capsid increased livertransduction and imparted heparin binding.

Data with AAV2 serotype vectors indicate that a single substitution oftyrosine to phenylalanine (Y→F) dramatically improves viral traffickingfrom endosome to the nucleolus by preventing capsid phosphorylation,subsequent ubiquitination and degradation via proteasome (Zhong et al.,2008). These studies have led to the generation of a number of vectorswith increased transduction efficiency in different cell types andtissues. Such vectors were used to improve F.IX gene transfer to murinehepatocytes for the phenotypic correction of hemophilia B (Markusic etal., 2011). These tyrosine-mutant AAV vectors also led to highefficiency transduction of mouse retina for the potential treatment ofocular diseases (Petrs-Zilva et al., 2009). Although AAV6 serotype hasshown higher transduction efficiency than AAV2 in dendritic cells (Veronet al., 2007; Taylor and Ussher, 2010), these studies have focused onAAV2 because these vectors have been studied more extensively in bothbasic research and clinical settings, however AAV6 vectors may bedeveloped with a similar strategy as described herein.

It has become abundantly clear that phosphorylation of surface-exposedtyrosine-residues on AAV2 capsids negatively impacts the transductionefficiency of these vectors, which can be dramatically augmented by theuse of specific inhibitors of cellular EGFR-PTK, known to phosphorylatethese residues (Zhong et al., 2008). In the present example, the role ofphosphorylation of serine residues in the life cycle of AAV2 vectors wasmore fully delineated.

Indeed, the transduction efficiency of both ssAAV and scAAV vectorscould be augmented by pre-treatment of cells with specific inhibitors ofJNK and p38 MAPK, implying that one or more surface-exposed serineand/threonine residues on the AAV2 capsid becomes phosphorylated insidethe host cell and that this modification is detrimental to capsidtrafficking to the nucleus.

Next, each of 15 surface-exposed serine residues was mutatedindividually, but only three of these mutations led to an increase intransduction efficiency in different cell types, which ranged from˜2-fold to ˜20-fold. However, unlike the tyrosine-mutants (Markusic etal., 2011), combining multiple mutations did not augment thetransduction efficiency of either the double-mutants (S458+662V andS492+662V), or the triple-mutant (S458+492+662V) AAV2 vectors in vitro.In this context, it is noteworthy that in a report by DiPrimio et al.,(DiPrimio et al., 2008), in which the HI loop located between the H andI strands of the conserved core β-barrel and contains residue S662 wascharacterized, both site-directed mutagenesis and peptide substitutionsshowed that this capsid region plays a crucial role in AAV capsidassembly and viral genome packaging (FIG. 22A and FIG. 22B) (Xie et al.,2002). Although the S662 residue was not specifically targeted in thosestudies, the transduction efficiency of most of these mutants was eitherunchanged, or was reduced by up to 27-fold. The HI loop, which formsinteractions between icosahedral five-fold symmetry related VPs and lieson the floor of the depression surrounding this axis, was also proposedto undergo a conformational re-arrangement that opens up the channellocated at the icosahedral fivefold axis following heparin binding byAAV2 (Levy et al., 2009). Residues S458 and 492 are located adjacent toeach other (contributed from symmetry related VPs) on the outer surfaceof the protrusions (surrounding the icosahedral three-fold axes) facingthe depression at the two-fold axes. Previous mutation of residuesadjacent to S458A, S492A and S492T had no effect on capsid assembly andresulted in no effect on transduction efficiency (Lochrie et al., 2006),which confirms the critical role that particular amino acids plays inpackaging efficiency and biological activity of AAV. Additionalstructural analyses of these data revealed the following: For the threemutants with low yields, the side-chain of the residues interact withmain-chain atoms from the same VP monomer, and S267V with a low titer,interacts with D269 from the same monomer. For another capsid mutant,S668V, which is located in the HI loop and shown to play a role incapsid assembly (DiPrimio et al., 2008), no obvious disruption ofinteraction was observed with the substitution. Interestingly, all ofthese residues, regardless of assembly phenotype, are at interfacepositions but only 458 and 492 involved in inter-VP interactions. Theother residues are only involved in intra-VP interactions, if any. Thus,it is possible that the changes in the no capsid or low capsid yieldmutants result in misfolding for their VPs or the abrogation offormation of multimers formation required for assembly when changed toalanine.

In the setting of tumor immunotherapy, the time of T cell activation andthe potency and longevity of CD8 T cell responses are crucial factors indetermining therapeutic outcome. Thus, the investors further evaluatedwhether increased transduction efficiency of moDC by the serine-mutantAAV2 vectors correlated with superior priming of T cells. Humantelomerase was used as a specific target since it has been shown innumerous studies and clinical trials to be an attractive candidate for abroadly expressed rejection antigen for many cancer patients (Harley,2008; Beatty and Vonderheide, 2008). These results suggest thatmodification of the AAV2 capsid might be beneficial in terms ofproducing more specific and effective vectors for gene delivery.

It is also important that one of the main obstacles, the induction ofimmuno-competition in cellular immune responses against vector-derivedand transgene-derived epitopes, can probably be overcome not only by thereplication-deficiency and lack of viral proteins expressed byrecombinant AAV2, but also the fact that less capsid of modified viralparticles will be degraded by host proteosomes and thus, provide lessmaterial for presentation.

Example 5 Optimization of the Capsid of rAAV2 Vectors

Adeno-associated virus (AAV) vectors are currently in use in a number ofPhase I/II clinical trials as delivery vehicles to target a variety oftissues to achieve sustained expression of therapeutic genes (Daya andBerns 2008; Mueller and Flotte 2008; Srivastava 2008; Asokan et al.,2012; Flotte et al., 2012). However, large vector doses are needed toachieve therapeutic benefits. The requirements for sufficient amounts ofthe vector pose a production challenge, as well as the risk ofinitiating the host immune response to the vector (High and Aubourg,2011; Mendell et al., 2012, Mingozzi and High, 2011). More specifically,recombinant vectors based on AAV2 serotype were initially used in aclinical trial for the potential gene therapy of hemophilia B, but inthis trial, therapeutic level of expression of human Factor IX (hF.IX)was not achieved at lower vector doses, and at higher vector doses, thetherapeutic level of expression of hF.IX was short-lived due to acytotoxic T cell (CTL) response against AAV2 capsids (Manno et al.,2006; Mingozzi and High, 2007; Mingozzi et al., 2007).

In a more recent trial with recombinant vectors based on AAV8 serotype,therapeutic levels of expression of hF.IX were been achieved, but animmune response to AAV8 capsid proteins was observed (Aslanidi et al.,2012). Thus, it is critical to develop novel AAV vectors with hightransduction efficiency that can be used at lower doses. Cellularepidermal growth factor receptor protein tyrosine kinase (EGFR-PTK)negatively affects transgene expression from recombinant AAV2 vectorsprimarily due to phosphorylation of AAV2 capsids at tyrosine residues,and tyrosine-phosphorylated capsids are subsequently degraded by thehost proteasome machinery (Zhong et al., 2008; Markusic et al., 2010).Selective inhibitors of JNK and p38 MAPK serine/threonine kinases alsoimproved the transduction efficiency of AAV2 vectors, suggesting thatphosphorylation of certain surface-exposed serine and/or threonineresidues might also decrease the transduction efficiency of thesevectors. These studies led to the development of tyrosine- andserine-mutant AAV2 vectors, which has been shown to transduce variouscell types with significantly higher efficiency than the WT vectors.(Aslanidi et al., 2012; Zhong et al., 2008; Markusic et al., 2010;Petrs-Silva et al., 2009) In addition to the tyrosine and serineresidues, the elimination of surface-exposed threonine residues bysite-directed mutagenesis also led to an increase in the transductionefficiency at lower vector doses. In this example, each of the 17surface-exposed threonine residues was substituted with valine (V)residues by site-directed mutagenesis, and four of these mutants, T455V,T491V, T550V, T659V, were shown to increase the transduction efficiencybetween fold in human HEK293 cells. Because the tyrosine triple-mutant(Y730F+500+444F) vector transduced murine hepatocytes most efficientlythan WT (Aslanidi et al., 2012; Zhong et al., 2008; Markusic et al.,2010; Petrs-Silva et al., 2009), these mutations were subsequentlycombined with the best-performing single serine-mutant (S662V) andsingle threonine-mutant (T491V) to generate the following vectors: twoquadruple (Y444+500+730F+S662V; Y730+500+44F+T491V) and one quintuple(Y444+500+730F+S662V+T491V). The quadruple-mutant (Y444+500+730F+T491V)vector efficiently transduced a murine hepatocyte cell line in vitro aswell as primary murine hepatocytes in vivo at reduced doses, whichimplicated the use of these vectors in human gene therapy in general,and hemophilia in particular.

Materials and Methods

Cells.

Human embryonic kidney cell line, HEK293, and murine hepatocyte cellline, H2.35, cells were obtained from the American Type CultureCollection (Manassas, Va., USA), and maintained as monolayer cultures inDMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Sigma)and antibiotics (Lonza).

Production of Recombinant Vectors.

Recombinant AAV2 vectors containing either EGFP (scAAV2-GFP) or fireflyluciferase gene (Flue) (ssAAV2-Fluc) driven by the chicken β-actinpromoter (CBA) were generated as described previously (Aslanidi et al.,2012; Aslanidi et al., 2009; Zolotukhin et al., 2002; Kohlbrenner etal., 2005). Briefly, HEK293 cells were transfected usingpolyethylenimine (PEI, linear, MW 25,000, Polysciences, Inc.).Seventy-two hrs' post-transfection, cells were harvested and vectorswere purified by iodixanol (Sigma) gradient centrifugation and ionexchange column chromatography (HiTrap Sp Hp 5 mL, GE Healthcare). Viruswas then concentrated and buffer exchanged into Lactated Ringer'ssolution in three cycles using centrifugal spin concentrators (Apollo,150-kDa cut-off, 20-mL capacity, CLP). To determine genome titers, tenIA of purified virus were incubated with DNase I (Invitrogen) at 37° C.for 2 hr, then with Proteinase K (Invitrogen) at 55° C. for anadditional 2 hr. The reaction mixture was purified by phenol/chloroform,followed by chloroform extraction. Packaged DNA was precipitated O/Nwith ethanol in the presence of 20 μg glycogen (Invitrogen). DNaseI-resistant AAV2 particle titers were determined by qPCR with thefollowing primer-pairs specific for the CBA promoter:

Forward: (SEQ ID NO: 20) 5′-TCCCATAGTAACGCCAATAGG-3′, Reverse:(SEQ ID NO: 21) 5′-CTTGGCATATGATACACTTGATG-3′,

and SYBR GreenER PCR Master Mix (Invitrogen) (Aslanidi et al., 2012;Aslanidi et al., 2009).

Site-Directed Mutagenesis.

A two-stage PCR was performed with plasmid pACG2 as described previously(Aslanidi et al., 2012; Wang and Malcolm, 1999) using Turbo PfuPolymerase (Stratagene). Briefly, in stage one, two PCR extensionreactions were performed in separate tubes for the forward and reversePCR primers for 3 cycles. In stage two, the two reactions were mixed anda PCR reaction was performed for an additional 15 cycles, followed byDpnI digestion for 1 hr. Primers were designed to introduce changes fromthreonine (ACA) to valine (GTA) for each of the residues mutated.

Recombinant AAV Vector Transduction Assays In Vitro.

Human HEK293 were transduced with 1×10³ vgs/cell, and murine hepatocytesH2.35 cells were transduced with 2×10³ vgs/cell with WT and mutantscAAV2-GFP vectors, respectively, and incubated for 48 hr. Transgeneexpression was assessed as the total area of green fluorescence (pixel2)per visual field (mean±SD) as described previously (Aslanidi et al.,2012; Zhong et al., 2008; Markusic et al., 2010). Analysis of variancewas used to compare test results and the control, which were determinedto be statistically significant.

Analysis of Vector Genome Distribution in Cytoplasm and NuclearFractions.

Approximately 1×10⁶ H2.35 cells were infected by either WT or mutantscAAV2-GFP vectors with MOI 1×10⁴ vgs/cell. Cells were collected atvarious time points by trypsin treatment to remove any adsorbed andun-adsorbed viral particles and then washed extensively with PBS.Nuclear and cytoplasmic fractions were separated with Nuclear andCytoplasmic Extraction Reagents kit (Thermo Scientific) according tomanufacturer instruction. Viral genome was extracted and detected byqPCR analysis with the CBA specific primers described above. Thedifference in amount of viral genome between cytoplasmic and nuclearfractions was determined by the following rule: C_(T) values for eachsample from cells treated with virus were normalized to correspondingC_(T) from mock treated cells (ΔC_(T)). For each pairwise set ofsamples, fold change in packaged genome presence was calculated as foldchange=2^(−(ΔCT-cytoplasm-ΔCT-nucleus)). Data from three independentexperiments were presented as a percentage of the total amount ofpackaged genome in the nuclear and cytoplasmic fractions.

In Vivo Bioluminescence Imaging.

All animal experiments were performed per institutional policies, andall procedures were done in accordance with the principles of theNational Research Council's Guide for the Care and Use of LaboratoryAnimals. All efforts were made to minimize suffering. Ten-week-oldC57BL/6 male mice (Jackson Laboratory, Bar Harbor, Me.) were injectedintravenously with 1×10¹⁰ vgs/animal of WT and mutant ssAAV2-Flucvectors (n=3). Luciferase activity was analyzed two weeks post injectionusing a Xenogen IVIS Lumina System (Caliper Life Sciences). Briefly,mice were anesthetized with 2% isofluorane and injectedintraperitoneally with luciferin substrate (Beetle luciferin, CaliperLife Sciences) at a dose of 150 μg/g of body weight. Mice were placed ina light-tight chamber and images were collected at 5 min after thesubstrate injection. Images were analyzed by the Living Image 3.2software (Caliper Life Sciences) to determine relative signal intensity.

Visualization of the Position of the Mutant Residues on the AAV2 Capsid.

The atomic coordinates for the AAV2 VP3 crystal structure (residues 217to 735, VP1 numbering) (Protein Data Bank (PDB) accession no. 1lp3; [Xieet al., 2002]) was downloaded and used to generate a complete capsidmodel using the Oligomer generator application in VIPERdb (Carrillo-Tripet al., 2009). This generates 60 VP3 copies for creating the T=1icosahedral capsid via matrix multiplication. The structure was viewedwith the program COOT (Xie et al., 2002) and figures were generatedusing either of the computer programs, PyMOL (Schrodinger, LLC) andRIVEM (Xiao and Rossman, 2007).

Statistical Analysis.

Results are presented as mean±S.D. Differences between groups wereidentified using a grouped-unpaired two-tailed distribution of Student'st-test. P-values <0.05 were considered statistically significant.

Results

Site-Directed Mutagenesis of Surface-Exposed Threonine Residues on AAV2Capsid.

The AAV2 capsid contains 45 threonine (T) residues in the capsid viralprotein 3 (VP3) common region of the three capsid VPs, VP1, VP2, andVP3. Seventeen of these (251, 329, 330, 454, 455, 503, 550, 592, 581,597, 491, 671, 659, 660, 701, 713, 716) are surface-exposed. (Xie etal., 2002) Each of the 17 T residues was substituted with valine (V) bysite-directed mutagenesis as described previously (Aslanidi et al.,2012; Zhong et al., 2008). Most mutants could be generated at titerssimilar to the WT AAV2 vectors, with the exception of T329V and T330Vthat were produced at ˜10-fold lower titers, and T713V and T716V, whichproduced no detectable levels of DNase I-resistant vector particles.Each of the T-V mutant vectors was evaluated for transduction efficiencyin HEK293 cells. These results, shown in FIG. 26A and FIG. 26B, indicatethat of the 17 mutants, the T491V mutant transduced HEK293 cells ˜4-foldmore efficiently than its WT counterpart did. The transductionefficiency of the T455V, T550V, T659V mutant vectors were increased by˜2-fold. These data indicated that phosphorylation of specific tyrosine,serine, and threonine residues on AAV2 capsid by cellular kinases is acritical determinant of the transduction efficiency of these vectors.

Multiple Mutations of Surface-Exposed Threonine Residues Further ImproveTransduction Efficiency of AAV2 Vectors.

To evaluate whether the transduction efficiency of the threonine-mutantAAV2 vectors could be enhanced further, the following multiple-mutantvectors were generated: three double-mutants (T455+491V; T550+491V;T659+491V), two triple-mutants (T455+491+550V; T491+550+659V), and onequadruple-mutant (T455+491+550+659V). Each of the multiple-mutantvectors packaged genome titers similar to the WT AAV2 vectors. Inside-by-side comparisons, each of the multiple-mutant vectors was shownto transduce HEK293 more efficiently than the WT and thesingle-threonine mutant AAV2 vectors (FIG. 27A and FIG. 27B). The bestperforming vector was identified to be the triple-mutant(T491+550+659V), with the transduction efficiency ˜10-fold higher thanthe WT vector, and ˜3-fold higher than the best single-mutant (T491V)vector. These data confirmed that combining several threonine-mutationson a single viral capsid led to a synergetic effect in augmenting thetransduction efficiency.

Optimized Threonine-Mutant AAV2 Vectors Efficiently Transduce MurineHepatocytes In Vitro.

The tyrosine triple-mutant (Y444+550+730F) vector described in previousexamples has been shown to be efficient in transducing murinehepatocytes in a comparison of vectors containing up to 7 surfacetyrosine to phenylalanine changes (Markusic et al. 2010; Jayandharan etal., 2011). Thus, it was of interest to evaluate whether combining thebest performing single-serine (S662V) and single-threonine (T491V)mutations with the triple-tyrosine mutant could further increase thetransduction efficiency of these vectors to produce even furtherimproved expression vectors in accordance with the methods describedherein.

To that end, several multiple-mutants were generated as follows: twoquadruple (Y444+500+730F+T491V; Y444+500+730F+S662V), and one quintuple(Y444+500+730F+T491V+S662V) mutant vectors. Comparison of thetransduction efficiency of these mutants with the WT and the tyrosinetriple-mutant AAV2 vectors in H2.35 cells showed that the expressionlevel from the Y444+500+730F+T491V mutant was ˜2-3-fold higher than forthe tyrosine triple-mutant AAV2 vector, and ˜24-fold higher than the WTAAV2 vector (FIG. 28A and FIG. 28B). Interestingly, combining the S662Vmutation with the tyrosine triple-mutant vector, or with thetyrosine-threonine quadruple-mutant vector, negatively affected theirtransduction efficiency. Addition of several other threonine mutations,such as T550V and T659V, also did not augment the transductionefficiency of the Y444+500+730F+T491V quadruple-mutant AAV2 vector.Additional studies are warranted to gain a better understanding of thecomplex interactions among these surface-exposed Y, S, and T residues aswell as their phosphorylation status.

Multiple Mutations Enhance Intracellular Trafficking and NuclearTranslocation of AAV2 Vectors.

Prevention of phosphorylation of surface-exposed tyrosine residues onthe AAV2 capsid improved intracellular trafficking of tyrosine-mutantvectors and increases the number of the viral genomes translocated tothe nucleus (Zhong et al., 2008; Zhong et al., 2008). In this example,the addition of the T491V mutant to the tyrosine triple-mutant vectorwas assigned for its ability to augment this transduction efficiency byfurther increasing nuclear transport of these vectors. To this end, thekinetics of transgene expression in H2.35 cells mediated by theY444+500+730F+T491V quadruple-mutant were evaluated and compared to theY444+500+730F triple-mutant and the WT AAV2 vectors. These results areshown in FIG. 29A and FIG. 29B. As can be seen, EGFP expression from thetyrosine-threonine quadruple-mutant vector was ˜2-3fold higher at eachtested time point, and could be detected as early as 16 hrpost-infection. These results suggested that the early-onset oftransgene expression from the quadruple-mutant vectors could be due tomore efficient nuclear transport of these vectors. To test thispossibility experimentally, qPCR analysis was used to quantitate thevector genomes in cytoplasmic and nuclear fractions of H2.35 cellsinfected with the WT and the two mutant AAV2 vectors at different timepoints. The vector genome ratios in the two cellular fractions are shownin FIG. 30A and FIG. 30B. Whereas ˜20% of the genomes from the WT AAV2vectors, and ˜45% of the genomes from the triple-mutant vectors weredetected in the nuclear fraction 16 hr post-infection, more than 70% ofthe vector genomes from the quadruple-mutant were detected at the sametime-point. Similarly, only ˜45% of the genomes from the WT AAV2 vectorswere detected in the nuclear fraction 48 hr post-infection, ˜80% of thegenomes from the triple-mutant vectors, and ˜90% of the vector genomesfrom the quadruple-mutant were detected in the nuclear fraction at thesame time-point. Thus, these data corroborated the hypothesis thatcombining the threonine (T491V) mutation with the tyrosine triple-mutant(Y444+500+730F) vector leads to a modest improvement in the nucleartranslocation of these vectors, which correlated with a faster onset ofgene expression and the observed improvement in the transductionefficiency.

Optimized AAV2 Vectors are Highly Efficient in Transducing MurineHepatocytes In Vivo.

The transduction efficiency of the optimized AAV2 vectors was evaluatedin a murine model in vivo. Each of multiple-mutant vectors was packagedwith a single-stranded firefly luciferase (Flue) AAV2 genome, and˜1×10¹⁰ vgs of each vectors were injected intravenously into C57BL/6mice (n=3 for each group). Levels of expression of Fluc gene, assessedtwo weeks post-injection by bioluminescence imaging, showed thatexpression from the Y444+500+730F+T491V quadruple-mutant vector was˜3-fold higher than that from the tyrosine triple-mutant vector. Onerepresentative animal from each group and the quantification of thesedata are presented in FIG. 31A and FIG. 31B. Consistent with the dataobtained in vitro, the addition of S662V mutation had a negative effecton the transduction efficiency of both the tyrosine-triple-mutant andthe tyrosine-threonine quadruple-mutant vectors. Exemplary single andmultiple-mutation capsid proteins of the present invention include, butare not limited to, those illustrated in Table 5:

TABLE 5 SUMMARY OF EXEMPLARY MUTATIONS OF SURFACE- EXPOSED TYROSINE (Y),SERINE (S), AND THREONINE (T) RESIDUES ON THE AAV2 CAPSID SingleMutations Double Mutations Triple Mutations Multiple Mutations Y252FY252F + Y730F Y444 + 500 + 730F Y272 + 444 + 500 + 730F Y272F Y272F +Y730F Y730F + S662V + T491V Y272 + 444 + 500 + 730F Y444F Y444F + Y730FS458 + 492 + 662V Y272 + 444 + 500 + 730F Y500F Y500F + Y730F T455 +550 + 491V Y272 + 444 + 500 + 700 + 730F Y700F Y700F + Y730F T550 +659 + 491V Y272 + 444 + 500 + 704 + 730F Y704F Y704F + Y730F Y252 +272 + 444 + 500 + 704 + 730F Y730F Y444F + T550F Y272 + 444 + 500 +700 + 704 + 730F S261V S458V + S492V Y252 + 272 + 444 + 500 + 700 +704 + 730F S264V S458V + S662V Y444 + 500 + 730F + T491V S267V S492V +S662V Y444 + 500 + 730F + S458V S276V T455 + T491V Y444 + 500 + 730F +S662V + T491V S384V T550 + T491V Y444 + 500 + 730F + T550 + T491V S458VT659 + T491V Y444 + 500 + 730F + T659 + T491V S468V T671 + T491V S492VY730F + T491V S498V S662V + T491V S578V Y730F + S662V S658V S662V S662AS662D S662F S662H S662N S662L S662I S668V S707V S721V T251V T329V T330VT454V T455V T491V T503V T550V T592V T597V T581V T671V T659V T660V T701VT713V T716V The first letter corresponds to the amino acid in thewild-type AAV2 capsid, the number is the VP3 amino acid position thatwas mutated, and the last letter is the mutant amino acid.

Discussion

Recombinant AAV-based vectors are attractive delivery vehicles for genereplacement therapy as a potential treatment for a variety of geneticdisorders. Although AAV vectors have been used successfully in manyanimal models, and recently shown efficacy in several clinical trials, anumber of steps in the life cycle of AAV continue to appear to limit theeffectiveness of these vectors in gene therapy. Some of these stepsinclude intracellular trafficking, nuclear transport, uncoating, andviral second-strand DNA synthesis (Ding et al., 2005; Harbison et al.,2005; Nonnenmacher and Weber, 2012).

The simple organization and natural plasticity of AAV structural andregulatory components provide a unique opportunity to manipulate theviral capsid and the genome to develop customized recombinant vectorswith distinctive features. Significant progress has been made in thepast decade to improve the specificity and the transduction efficiencyof recombinant AAV vectors. For example, specific mutations in the viralinverted terminal repeat (ITR) sequences have led to development ofself-complementary AAV (scAAV) vectors, which overcome the rate-limitingstep of viral second-strand DNA synthesis, and dramatically increasetransgene expression levels in various types of the cells and tissues(McCarty et al., 2003; Wang et al., 2003). Additional studies on capsidstructure analyses, combined with a wealth of information emanating frommutagenesis studies on the capsid genes, have led to the identificationof specific regions which play a critical role in vector encapsidation,tissue-tropism, and intracellular trafficking of these vectors (Lochireet al., 2006; Muzyczka and Warrington, 2005; Wu et al., 2006; Gao etal., 2003; Vandenberghe et al., 2009; Wu et al., 2006).

In the previous examples, it was shown that substitution ofsurface-exposed specific tyrosine (Y) and serine (S) residues on AAV2capsids significantly increased the transduction efficiency of thesevectors, both in vitro and in vivo, presumably by preventingphosphorylation, subsequent ubiquitination, and proteasome-mediateddegradation. Since surface-exposed specific threonine (T) residues onAAV2 capsids would likewise be expected to undergo phosphorylation, inthis example each of the 17 surface-exposed T residues weresystematically mutagenized, and several single-mutant vectors wereidentified that could increase the transduction efficiency up to 4-fold.Combinations of multiple T mutations on a single capsid identifiedmodifications that further augmented the transduction efficiency up to˜10-fold, compared with that of the WT AAV2 vector in HEK293 cells.

Two independent groups have previously reported mutations of specific Tresidues on AAV2 capsids. For example, Lochrie et al., 2006, targetedthe T residues at positions 330, 454, 455, 491, 503, and 550 in a tourde force effort to identify surface regions that bind antibodies, andDiPrimio et al. (2008), targeted the T residue at position 659 in aneffort to identify regions critical for capsid assembly and genomepackaging. In both studies, the T residues were substituted with eitheralanine (A), serine (S), or lysine (K) residues, or by peptidesubstitution. However, no increase in the transduction efficiency of anyof the mutant vectors was observed. In contrast, in the example, thesurface-exposed T residues were substituted with valine residues. Thisfurther corroborates the recent observation for the critical role playedby specific amino acid type in modulating the biological activity of AAVvectors (Aslanidi et al., 2012; Li et al., 2012).

When the most efficient threonine-mutation (T491 V) was combined with apreviously reported tyrosine triple-mutation (Y444+500+730F) (Markusicet al. 2010) to generate a Y-T quadruple-mutant (Y444+500+730F+T491V)vector, the transduction efficiency of this vector was ˜2-3-fold higherthan the tyrosine triple-mutant vector in murine hepatocytes, both invitro and in vivo. However, combining the most efficient S-mutation(S662V) (Aslanidi et al., 2012) with the tyrosine triple-mutationnegatively affected the transduction efficiency of the Y-S quadruplemutant (Y444+500+730F+S662V) vector aswell as the Y-S-T pentuple mutant(Y444+500+730F+S662V+T491V) vector. Although several other combinationsshowed greater transduction efficiency compared with the WT AAV2 vector,neither combination of similar (quadruple, pentuple orsextuple-tyrosine; and triple and quadruple-threonine mutants), norcombination of the best performing YST mutations reached the level ofexpression from the triple-tyrosine mutant vector. In view of the largenumber of combinations of mutations tested, only the mutations thatsignificantly increased the transduction efficiency over thetriple-tyrosine mutant vector were characterized in detail here.

The 17 AAV2 surface-exposed threonine residues are scattered throughoutthe capsid. Four of the mutations (T329V, T330V, T713V, and T716V)resulted in significant defects in assembly and vector production, andthey could not be further characterized. Residues 329 and 330 arelocated in the α-surface loop (DE loop) located between the βD and βEstrands of the core β-barrel of the AAV2 VP3 structure (Xie et al.,2002). Five of these loops, from icosahedral five-fold symmetry relatedVP3s assembly a channel at this axis which connects the interior andexterior surfaces of the capsid (FIG. 32A). As was observed in aprevious study (Bleker et al., 2006), titers for these mutants weresignificantly reduced consistent with a role for the channel in genomepackaging. Residues 713 and 716 are located on the wall/raised capsidregion between the depressions at and surrounding the icosahedral two-and five-fold axes, respectively (FIG. 32A and FIG. 32B). Theirside-chains participate in polar interactions with symmetry related VP3monomers and it is likely that mutation results in a defect in capsidassembly. A role in capsid assembly for residues located at theicosahedral two-fold axis is consistent with a recent report in whichthey observe that the AAV2 residues that mediated the interaction withthe assembly-activating protein (AAP) were located at this capsid region(Naumer et al., 2012).

Residues T455, T491, T550, and T659, showing an increased transductionphenotype when mutated to valine or alanine, are located on theprotrusions which surround the icosahedral three-fold axis (T455, T491,and T550) or on the HI loop (between βH and βI of the core β-barrel)(T659) which is lies on the depression surrounding the channel at theicosahedral five-fold axis of the AAV2 capsid. The residues on theprotrusion, a prominent feature on the capsid assembled from two VP3monomers, are located close to the top (455), side facing the two-folddepression (491), and side facing the depression surrounding thefive-fold (550), respectively, of the protrusions. This AAV regioncontains the most variability in sequence and structure, and with theexception of residue 659, the other three threonine residues are locatedto define VP3 variable regions (VRs) (Govindasamy et al., 2006). Alongwith T659, these residues form a footprint on the capsid surface thatextends over the top of the protrusion towards the depressionsurrounding the icosahedral five-fold axis (FIG. 32A and FIG. 32B).Their surface exposure is consistent with the potential to interact withhost molecules, which could include kinases. Interestingly, thisfootprint is flanked by the residues in the triple-tyrosine mutant,Y444, Y500, and Y730, with T491 located proximal to tyrosine residueY730 in a depiction of the capsid surface amino acids (FIG. 32B). Thisresidue, which sits in the depression at the icosahedral axis of thecapsid, showed the highest increase in transduction compared to WT AAV2when of the seven surface-exposed tyrosines where mutated to phenylanineresidues (Zhong et al. 2008). Significantly, the two-fold capsid regionis observed to undergo pH-mediated structural transitions when thehomologous AAV8 was examined at the conditions encountered duringtrafficking in the endocytic pathway (Nam et al., 2011). It is possiblethat the mutations of the AAV2 improve transduction efficiency throughaltered receptor binding mechanisms. Residues mediating AAV2 and AAV6interaction with heparan sulfate receptors, R585 and R588, and K531(structurally equivalent to E530 in AAV2), respectively, are close tothis foot (FIG. 26B), and residues 491 and 500, in VRV, are located inone of two large regions on the surface of the AAV2 capsid that has beenimplicated in binding to the LamR receptor in AAV8 (Akache et al.,2006). Amino acids in VRV also play a role in the AAV9 capsid binding toits glycan receptor, galactose.

The decreased transduction efficiency phenotype of the mutantscontaining the S662V mutations is difficult to explain given thelocation of this residue within the footprint delineated by the residueswhich enhance transduction when mutated to eliminate potentialphosphorylation (FIG. 32A and FIG. 32B). In addition, it has been shownthat a mutation of this residue to valine improved transduction relativeto WT AAV2 (Aslanidi et al., 2012). Residue S662, like T659, is locatedin the HI loop that extends over adjacent five-fold symmetry related VP3monomers and likely plays a role in stabilizing the pentameric subunits.However, the serine side-chain is not engaged in any inter- orintra-subunit interactions, and while the HI loop has been reported tobe a determinant of capsid assembly and genome packaging (DiPrimio etal., 2008), it tolerated single amino acid substitution (Aslanidi etal., 2012). Thus, its effect is likely due to the abrogation of a capsidinteraction utilizing the footprint containing the triple-tyrosinemutant residues and T491. Significantly, the phenotypes for mutations innearby amino acids that make up the HI loop, for example, amino acidresidue 664, substituting either serine (mut45subSer14) or a FLAGepitope (mut45SubFLAG10), were non-infectious or not assembled intoviral capsid (Wu et al., 2000). However, an HA insertion at the sameposition produced capsids that were partially defective, yet still boundheparin (Wu et al., 2000).

Whereas only ˜45% of the vector genomes delivered by the WT AAV2 vectorswere present in the nucleus at 48 h post infection, >90% of the vectorgenomes delivered by the Y-T quadruple-mutant vector were present at thesame time point. This indicates improved trafficking kinetics for themutant that would be consistent with reduced re-direction to theproteasome. The modest (˜2-3-fold) increase in the transductionefficiency of these vectors compared to the tyrosine triple-mutantvectors is also consistent with the ˜10% increase in nuclear vectorgenome delivery, i.e. ˜90% compared to ˜80%.

The various combinations of surface tyrosine, serine, and threoninemodifications clearly showed that there is an optimal combination toachieve maximal augmentation. These studies also highlighted therequirement for specific residue types in AAV interactions duringinfection and for enhancing transduction. It is possible that theindividual mutations, which did not show a significant increase in thetransduction efficiency as single changes, can form superior vectorswhen combined in a single capsid.

TABLE 6 COMPARISON OF TYROSINE RESIDUES IN AAV SEROTYPES (Surfaceexposed residues are shown with an “*” following their amino acidposition) AAV1 AAV* AAV* AAV4 AAV* AAV* AAV* AAV* AAV9 AAV* AAV* AAV

Y6 Y6 Y6 Y5 NA Y6 Y6 Y6 Y6 Y6 Y6 Y6 Y50 Y50 Y50 Y49 Y49 Y50 Y50 Y50 Y50Y50 Y50 Y50 Y52 Y52 Y52 Y51 Y51 Y52 Y52 Y52 Y52 Y52 Y52 Y52 Y79 Y79 Y79Y78 Y78 Y79 Y79 Y79 Y79 Y79 Y79 Y79 Y90 Y90 Y90 Y89 Y89 Y90 Y90 Y90 Y90Y90 Y90 Y90 Y93 Y93 Y93 Y92 Y92 Y93 Y93 Y93 Y93 Y93 Y93 Y93 Y252 Y252*Y252* Y246* Y242* Y252* Y253* Y253* Y252* Y253* Y246* Y255* Y257 Y257Y257 Y251 Y247 Y257 Y258 Y258 Y257 Y258 Y251 Y260 Y273* Y272* Y272*Y263* Y263* Y273* Y274* Y275* Y274* Y275* Y263* Y272* Y276 Y275 Y275 NAY266 Y276 Y277 Y278 Y277 Y278 NA NA Y282 Y281 Y281 Y272 Y272 Y282 Y283Y284 Y283 Y284 Y272 Y281 NA NA NA NA Y294 NA NA NA NA NA NA NA Y349 Y348Y348 Y339 Y339 Y349 Y350 Y351 Y350 Y351 Y339 Y348 Y353 Y352 Y352 Y343Y343 Y353 Y354 Y355 Y354 Y355 Y343 Y352 Y376 Y375 Y375 Y366 Y366 Y376Y377 Y378 Y377 Y378 Y366 Y375 Y378 Y377 Y377 Y368 Y368 Y378 Y379 Y380Y379 Y380 Y368 Y377 Y394 Y393 Y393 Y387 NA Y394 Y395 Y396 Y395 Y396 Y386Y395 Y398 Y397 Y397 Y391 Y390 Y398 Y399 Y400 Y399 Y400 Y390 Y399 Y414Y413 Y413 Y407 Y406 Y414 Y415 Y416 Y415 Y416 Y406 Y415 Y425 Y424 Y424Y418 NA Y425 Y426 Y427 Y426 Y427 Y417 Y426 Y442* Y441* Y441* Y435* Y434*Y442* Y443* Y444* Y443* Y444* Y434* Y443* NA NA NA NA Y436 NA NA NA NANA NA NA Y444* Y443* Y443* NA NA Y444* Y445* Y446* Y445* Y446* NA NAY445* Y444* Y444* NA NA Y445* Y446* Y447* Y446* Y447* NA NA NA NA NA NANA NA NA NA NA NA NA Y465 NA NA NA NA NA NA Y466 NA NA NA NA NA NA NA NANA Y457 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Y475 NA NANA NA NA NA NA NA NA NA Y467 Y476 NA NA NA NA Y461 NA NA NA NA NA NA NANA NA NA NA NA NA NA NA Y478 NA NA NA Y484 Y483 Y484 NA NA Y484 NA Y486Y484 Y486 NA NA NA NA NA Y491* NA NA NA NA NA NA Y490* Y499* NA Y500* NANA NA NA NA NA NA NA NA NA NA NA NA Y504* NA NA NA NA NA NA Y503* Y512*Y509 Y508 Y509 NA NA Y509 Y511 Y511 NA Y511 Y507 NA NA NA NA NA Y502 NANA NA NA NA NA NA NA NA NA NA Y521* NA NA NA NA NA NA NA NA NA NA NAY542* NA NA Y557* NA Y557* NA NA NA NA NA NA Y563* NA NA NA NA NA NA NANA Y576 Y577 NA NA NA Y578 Y579 Y577 Y579 NA NA NA NA NA NA Y585* NA NANA NA NA NA NA Y613* Y612* Y613* Y611* Y602* Y613* Y614* Y615* Y613*Y615* Y610* Y619* NA NA NA Y612 NA NA NA NA NA NA Y611 Y620 Y674* Y673*Y674* Y672* Y662* Y674* Y675* Y676* Y674* Y676* Y671* Y680* Y701 Y700*Y701 NA Y689* Y701* Y702* Y703* Y701* Y703* NA NA Y705* Y704* Y705*Y703* Y693* Y705* NA Y707* Y705* Y707* Y702* Y711* NA NA NA NA NA NA NAY708 Y706 Y708 NA NA Y721 Y720 Y721 Y719 Y709 Y721 Y722 Y723 Y721 Y723Y717 Y727 Y731* Y730* Y731* Y729* Y719* Y731* Y732* Y733* Y731* Y733*Y728* NA

indicates data missing or illegible when filed

TABLE 7 COMPARISON OF LYSINE RESIDUES IN AAV SEROTYPES (Surface exposedresidues are shown with an “*” following their amino acid position) AAV1AAV2 AAV3 AAV4 AAV5 AAV6 AAV7 AAV8 AAV9 AAV10 AAV11 AAV12 NA K24 NA NANA NA NA NA NA NA NA NA K26 K26 K26 NA NA K26 K26 K26 K26 K26 K26 K26K31 NA NA K30 K30 K31 K31 K31 NA K31 K31 NA K33 K33 K33 K32 K32 K33 K33K33 K33 K33 K33 K33 K38 NA NA NA NA K38 K38 K38 NA K38 K38 NA NA K39 NANA NA NA NA NA NA NA NA NA K51 K51 K51 K50 NA K51 K51 K51 K51 K51 K51K51 K61 K61 K61 K60 NA K61 K61 K61 K61 K61 K61 K61 K77 K77 K77 K76 NAK77 K77 K77 K77 K77 K77 K77 NA NA NA NA NA NA NA NA NA NA NA K81 K84 NAK84 K83 NA K84 K84 NA K84 K84 K84 NA NA K92 K92 K91 K91 NA NA NA K92 NANA K92 NA NA NA NA K102 NA NA NA NA NA NA NA NA K105 NA NA NA NA NA NAK105 NA NA NA NA NA NA NA K115 NA NA NA NA NA NA NA K122 K122 K122 K121K121 K122 K122 K122 K122 K122 K122 K122 K123 K123 K123 K122 K122 K123K123 K123 K123 K123 K123 K123 K137 K137 K137 NA K136 K137 K137 K137 K137K137 K137 K137 K142 K142 K142 K141 NA K142 K142 K142 K142 K142 K142 K142K143 K143 K143 K142 K142 K143 K143 K143 K143 K143 K143 K142 NA NA NA NANA NA NA NA NA NA NA K148 NA NA NA NA K150 NA NA NA NA NA NA NA NA NA NANA K152 NA NA NA NA NA NA NA NA NA NA NA K153 NA NA NA NA NA NA K160K161 K161 K161 K160 NA K161 K162 K162 K161 K162 K160 K164 NA NA NA K161NA NA K163 K163 NA K163 K161 K165 NA NA NA NA NA NA NA NA NA NA NA K166NA NA K164 K163 NA NA NA NA NA NA K163 NA NA NA NA NA K161 NA NA NA NANA NA K168 K168 NA NA K167 NA K168 NA NA K168 K169 NA NA K169 K169 K169K168 NA K169 K170 K170 K169 K170 K168 NA NA NA NA K169 NA NA NA NA NA NANA NA NA NA NA NA K232I NA NA NA NA NA NA NA K258* K258 K258* K252* NAK258* K259* K259* K258* K259* NA NA NA NA NA NA K251* NA NA NA NA NA NANA K310I K309 K309I K300I NA K310I K311I K312I K311I K312I K300I K309INA NA K310I NA NA NA K312I NA NA NA NA NA K315I K314 K314I K305I K305IK315I K316I K317I K316I K317I K305I K314I K322I K321 K321I K312I K312IK322I K323I K324I K323I K324I K312I K321I NA NA NA NA NA NA NA K333*K332* K333* NA NA NA NA NA NA NA NA NA NA NA NA NA K384I NA NA NA NAK394I NA NA NA NA NA NA NA NA NA NA K411I NA NA NA NA NA NA K410I K419INA NA NA NA K425I NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA K449* NANA NA K459I NA NA NA NA K459I NA NA NA NA NA NA NA NA NA NA NA NA NA NAK462* NA NA NA NA NA NA K459I K451I NA NA NA NA NA K458I K467I NA NA NAK469I NA NA NA NA NA NA NA NA K476I NA NA K470I K462I K476I K478I K478INA K478I K469I K478I NA NA NA K479I NA NA NA NA NA NA K478I K487I NA NANA NA NA NA NA NA NA NA NA K490I K491* K490 K491* K485* NA K491* K493*NA NA NA K484* K490 K493* NA NA NA NA K493* NA NA NA NA NA NA NA NA NAK492* NA NA NA NA NA NA K491* K493 NA NA NA K503* NA NA NA NA NA NAK502* K511 K508* K507 K508* NA NA K508* K510* K510* NA K510* NA NA K528*K527 K528* NA NA K528* K530* K530* K528* K530* NA NA NA NA NA NA NAK531* NA NA NA NA NA NA K533* K532 K533* NA NA K533* NA NA NA NA NA NANA NA NA K532* NA NA NA NA NA NA NA NA K545* K544 K545* NA NA K545*K547* K547* K545* K547* NA NA NA NA NA K544* NA NA NA NA NA NA NA NA NAK549 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA K553* NA NA NA NANA NA K556 NA NA NA NA NA NA K557* NA NA NA K567*- NA NA NA NA K567* NAK569* K567* K569* NA NA K621I K620 K621I K619I K610I K621I K622I K623IK621I K623I K618I K627I K641I K640 K641I K639I K630I K641I K642I K643IK641I K643I K638I K647I K650I K649 K650I K648I K639I K650I K651I K652IK650I K652I K647I K656I NA NA NA NA NA NA NA NA K664I NA NA NA K666*K665 K666* NA NA K666* K667* K668* K666* K668* NA NA NA NA NA NA K676INA NA NA NA NA NA NA K689I K688 K689I K687I K677I K689I K690I K691IK689I K691I K686I K695I K693I K692 K693I K691I K681I K693I K694I K695IK693I K695I K690I K699I K707* K706 K707* NA NA K707* K708* K709* K707*K709* NA NA NA NA NA K718I NA NA NA NA NA NA K717I NA Residues in boldare surface-associated lysines = * Resides that are located on theinterior of the capsid = I No homologous lysine at that position forthat serotype = NA

Residues not visible in the crystal structure of AAVs are shaded ingrey; however, biochemical data suggests that these amino acids arelocated inside the AAV capsid until some point in the virus life cyclewhen they are then externalized.

Example 6 Suppression of Human Liver Tumorigenesis by AAV3 Vectors

Hepatocellular carcinoma (HCC) ranks fifth among solid cancers with˜695,900 deaths worldwide each year (Thomas and Zhu, 2005; Jemal et al.,2011). During the past two decades, the incidence of HCC in the USA hastripled, while the 5-year survival rate has remained below 12%(El-Serag, 2011). It is even worse in Asia and Africa, with an annualincidence of 1 per 3,000 in China (Chen, et al., 2013). Currently,staging of HCC is considered crucial for planning of optimal therapy(Bruix et al., 2005). Patients with early HCC may benefit from radical(curative) therapies and those with intermediate stage may benefit frompalliative treatments. However, relapse is a frequent complication andtreatment failure rates are high. For those with advanced HCC,unfortunately, best supportive care is the only option (Verslype et al.,2009). Although there is one medicine, Sorafenib, approved by the U.S.Food and Drug Administration for advanced HCC, in a large Phase IIIclinical trial the median survival rate increased only from 7.9 to 10.7months (Llovet et al., 2008).

AAV vectors have shown remarkable efficacy in the treatment of Leber'scongenital amaurosis (Bainbridge et al., 2008; Maguire et al., 2008;Cideciyan et al., 2008; Cideciyan, 2010) hemophilia B (Nathwani et al.,2011) and aromatic L-amino acid carboxylase deficiency (Hwu et al.,2012). Glybera, a rAAV1 vector to treat lipoprotein lipase deficiency,is the first gene therapy product in the Western world (Melchiorri etal., 2013). In the early 2000s, conventional, single-stranded (ss) rAAV2vectors were used by investigators to target HCC in vivo (Su et al.,2000). Unfortunately, since the transduction efficiency of ssAAV2vectors is low, no transduction was observed in tumors larger than 2-mmvia systemic administration (Peng et al., 2000). More recently, deliveryof specific miRNAs in a mouse endogenous HCC model using rAAV8 vectorswas shown to result in inhibition of cancer cell proliferation (Kota etal., 2009; Hsu et al., 2012). However, rAAV8 vectors have a broadtropism to normal tissues other than the liver in murine models(Zincarelli et al., 2008; Gao et al., 2002; Wang et al., 2005) and innon-human primates (Nathwani, et al., 2006; Nathwani et al., 2007).

Interestingly, the inventors have demonstrated that rAAV3 vectors, whichfail to efficiently transduce any normal murine tissue in vivo,(Zincarelli et al., 2008; Palomeque et al., 2007; Markakis et al., 2010;Ling et al., 2010) were shown to transduce human HCC cells highlyefficiently both in vitro (Ling et al., 2010; Glushakova et al., 2009)and in vivo (Cheng et al., 2012). Although rAAV3 vectors also transduceprimary human hepatocytes, the transgene expression could be restrictedto malignant cells by using a HCC-specific promoter, α-fetoproteinpromoter (AFPp). In subsequent studies, the inventors observed thatrAAV3 vectors utilize the human hepatocyte growth factor receptor(hHGFR, also named c-Met) as a cellular co-receptor, (Ling et al., 2010)which indicates an opportunity to exploit rAAV3-based vectors intargeting human liver cancers, since hHGFR is over-expressed in most HCCcells (You et al., 2011). Furthermore, since AAV3 has lower incidence ofpre-existing neutralizing antibodies in humans compared with othercommonly used AAV serotypes (van der Marel et al., 2011), it has thepotential to be developed as a selective viral vector for gene therapyof human liver cancers. The present example demonstrates that furtheraugmentation of rAAV3-mediated transduction efficiency in human livercancer cells can be achieved through the elimination of specificsurface-exposed tyrosine (Y), serine (S), and threonine (T) residues onthe viral capsids. No observed significant alteration in cellularreceptor interaction in vitro and viral-tropism in vivo was associatedwith these modifications. Furthermore, significant inhibition oftumorigenesis in a human liver cancer xenograft model was achievedthrough systemic administration of optimized rAAV3 vectors carrying anovel therapeutic gene from traditional Chinese medicine, Trichosanthin.

Materials and Methods

Chemicals, Plasmids and Primers.

Grade I-A heparin sodium salt were purchased from Sigma-Aldrich.Recombinant hHGF was purchased from Life Technologies. Plasmid pHelperwas purchased from Agilent Technologies. Plasmids pdsAAV-CBAp-EGFP andpAAV-CBAp-FLuc were obtained from Dr. Xiao Xiao, University of NorthCarolina at Chapel Hill. Plasmid pdsAAV-AFPp-EGFP has been previouslydescribed (Glushakova et al., 2009). The TCS gene (Shaw et al., 1994)was synthesized by Life Technologies, based on the published sequence(T. kirilowii trichosanthin (TCS) mRNA, complete cds; GenBank:M34858.1), and sub-cloned into plasmid pdsAAV-AFPp-EGFP using AgeI andHindIII restriction sites. All plasmids were sequenced prior to use.

Construction of Optimized rAAV3 Capsid Plasmids.

A two-stage PCR procedure, using Turbo Pfu Polymerase (Stratagene) wasperformed to introduce site-specific mutations in the rAAV3 capsid, asdescribe previously (Zhong et al., 2008; Markusic et al., 2010; Aslanidiet al., 2013). Briefly, in stage one, two PCR extension reactions wereperformed in separate tubes for each mutant. One tube contained theforward PCR primer and the other contained the reverse primer. In stagetwo, the two reactions were mixed and a standard PCR mutagenesis assaywas carried out according to the manufacturer's instructions. All mutantplasmids were sequenced before use.

Cell Lines and Cultures.

Human hepatocellular carcinoma (Huh7) cells have been describedpreviously, (Ling et al., 2010; Cheng et al., 2012) and were maintainedin complete Dulbecco's modified Eagle medium (C-DMEM, Mediatech, Inc.)supplemented with 10% heat-inactivated fetal bovine serum (FBS,Sigma-Aldrich), 1% penicillin and streptomycin (Lonza). A newlyestablished human hepatoblastoma (Hep293TT) cell line, was obtained fromDr. Gail E. Tomlinson (University of Texas Health Science Center at SanAntonio) and was maintained in complete RPMI medium 1640 (LifeTechnologies) supplemented with 15% heat-inactivated FBS (Sigma-Aldrich)and 1% P/S (Lonza). Cells were grown as adherent culture in a humidifiedatmosphere at 37° C. in 5% CO₂ and were sub-cultured after treatmentwith trypsin-versene mixture (Lonza) for 2-5 min at room temperature,washed and re-suspended in complete medium.

rAAV Vectors Production.

Highly purified stocks of rAAV vectors were generated by thetriple-plasmid transfection protocol. Briefly, HEK293 cells wereco-transfected with three plasmids using Polyethelenimine (linear, MW25000, Polysciences, Inc.), and medium was replaced six hrspost-transfection. Cells were harvested 72-hrs' post-transfection,subjected to three rounds of freeze-thaw and then digested withBenzonase (Life Technology). Viral vectors were purified by iodixanol(Sigma-Aldrich) gradient ultra-centrifugation followed by ion exchangechromatography using HiTrap SP/Q HP (GE Healthcare), washed withphosphate-buffered saline (PBS, Mediatech, Inc.) and concentrated bycentrifugal spin concentrators with a 150 Kda molecular-weight cutoff(MWCO). The physical genomic titers of recombinant vector stocks weredetermined by quantitative DNA slot-blot and Southern blot analyses.

rAAV Vectors Transduction In Vitro.

Cells were seeded in 96-well plates at 5,000 or 10,000 cells per well inC-DMEM. Twenty-four hours later, cells were mock-treated or treated withindicated chemicals for 2 hrs. rAAV infections (MOI: 5×10³) were thenperformed in serum- and antibiotic-free DMEM medium for 2 hrs, with orwithout indicated chemicals, followed by extensive washes with PBS toremove the vector inoculum. Transgene expression was analyzed by eitherfluorescence microscopy or flow cytometry 72-hrs' post-transduction.

hHGF Competition Assay.

Huh7 cells were transduced with scAAV3-CBAp-EGFP vectors at an MOI of5×10³ vgs/cell. Vectors were premixed with increasing concentrations ofrecombinant hHGF. Cells were analyzed for EGFP expression levels 72 hrspost-transduction.

Animal Handling.

All animal experiments were approved by the appropriate regulatoryauthorities, and were performed according to specified guidelines foranimal care. Six- to ten-week old non-obese diabetic/severe combinedimmuno-deficient, IL2-gamma-deficient (NSG) male mice were purchasedfrom Jackson Laboratory.

Human Liver Cancer Xenograft Model.

Six- to ten-week old male NSG mice received subcutaneous injection of5×10⁶ Huh7 or Hep293TT cells on the ventral side of the neck betweenshoulder blades. Animals were kept in sterile cages until the end of theexperiment.

In Vivo FLuc Assays.

rAAV vectors were injected intravenously via tail-vein, or injecteddirectly into tumors in NSG mice. For in vivo FLuc imaging, mice wereweighed to calculate the volume of substrate, D-luciferin-K⁺ salt(Caliper Life Sciences), according to the dose of 4 mg/kg of body weightand anesthetized. The calculated volume of the 5 mg/mL of stocksubstrate solution was mixed with 100 μL of PBS and injectedintra-peritoneally. In vivo bioluminescence images were acquiredimmediately over a period of 5 min using a Xenogen machine equipped witha cooled couple-charged device camera (Xenogen). Signal intensity wasquantified using the camera control program, Living Image software andpresented as photons/second/cm²/steridian (p/s/cm²/sr).

Statistical Analysis.

Results are presented as mean±standard deviation (SD). Differencesbetween groups were identified using one-way ANOVA with Sidak's multiplecomparison test in GraphPad Prism 6 software.

Results

rAAV3 Vectors Selectively Transduce Human Liver Tumors In Vivo.

In the first set of studies, the transduction efficiency of rAAV3 andrAAV8 vectors in human HCC tumors was compared in a murine xenograftmodel in vivo. Non-obese, diabetic (NOD), severe-combinedimmune-deficient (scid), interleukin 2-deficient (IL2⁻) (NSG) mice, bothmale and female (n=4), were injected subcutaneously on the ventral sidebetween shoulder blades with 5×10⁶ human Huh7 cells, which formedtumors. Four-weeks later, mice were either mock injected, or injectedvia the tail vein with 1×10¹¹ viral genomes (vgs)/mouse of rAAV3 orrAAV8 vectors carrying the firefly luciferase gene (FLuc) under thecontrol of cytomegalovirus (CMV) enhancer/chicken β-actin hybridpromoter (CBAp) (FIG. 40A). Whole body bioluminescent imaging wasperformed 3 days post-vector administration. These results are shown inFIG. 36A. As can be seen, in both male and female mice injected withrAAV3 vectors, FLuc expression was restricted to the tumor, whereas inmice injected with rAAV8 vectors, FLuc expression was relatively morewidespread, but predominantly localized to liver, and the transductionefficiency of rAAV8 vectors was significantly higher in male mice, anobservation, consistent with previously published studies (Davidoff etal., 2003). Quantitative data further demonstrated that rAAV3vector-mediated transgene expression was restricted to tumors, andlittle transgene expression was detected in livers. In contrast, inrAAV8 vector-injected mice, only low-level transgene expression occurredin the tumors, whereas expression in the liver was significantly higher.To further corroborate these results, in the second set of experiments,rAAV3 and rAAV8 vectors were used in direct intra-tumor injections inmale NSG mice bearing human Huh7 tumors (n=4) at 1×10¹¹ vgs/mouse, andwhole body bioluminescence images were obtained 3 days post-vectorinjections. As can be seen in FIG. 36C, high-level transgene expressionwas localized to the tumor in mice injected with rAAV3 vectors, whereasin addition to the tumor, significant transgene expression in the liverwas also detected in mice injected with rAAV8 vectors. Thus, in contrastto rAAV8 vectors, rAAV3 vectors possess human liver tumor-tropism inthis experimental model in vivo.

In preliminary experiments, a diminution in transgene expression wasalso noted in tumors 5-days' post-vector administration, which was dueto the rapid growth of HCC tumors. Thus, to further corroborate theseresults, in the next set of experiments, direct intra-tumor injectionswas performed of rAAV3 and rAAV8 vectors at low (L=1×10¹¹ vgs/mouse) andhigh (H=1×10¹² vgs/mouse) doses. Whole-body bioluminescence imaging dataobtained at both day 3 and day 7 are shown in FIG. 36D. It was evidentthat, even at a high dose, ectopic expression in the liver in rAAV3vector-injected mice was minimal, whereas intra-tumor injection of rAAV8vectors resulted in strong transgene expression in the liver in a dose-and time-dependent manner. At Day 7, post-vector injections, mice weresacrificed and the viral genome copy numbers persisting in the livertissue samples were compared. These results, shown in FIG. 36E,indicated that a large amount of rAAV8 vector genomes were present inthe liver, whereas the numbers of rAAV3 vector genomes were minimal,corroborating earlier results. These studies, together with earlierresults (Ling et al., 2010; Glushakova et al., 2009; Cheng et al., 2012;Ling et al., 2011) provide a clear rationale for employing rAAV3vector-mediated gene therapy for HCC.

Further augmentation of rAAV3 vector-mediated transgene expression inhuman liver cancer cells in vitro can be achieved through modificationsof viral capsids. To further enhance the transduction efficiency ofrAAV3 vectors, site-directed mutagenesis of rAAV3 capsids was performed.In addition to mutagenesis of surface-exposed tyrosine (Y) tophenylalanine (F) residues (Cheng et al., 2012), surface-exposed serine(S), threonine (T), and lysine (K) residues were also mutagenized tovaline (V) and glutamic acid (E) or arginine (R) residues, respectively.Priority was given to the positions that are conserved among various AAVserotypes, and have previously been shown to augment the transductionefficiency of rAAV2 vectors (Zhong et al., 2008; Markusic et al., 2010;Aslanidi et al., 2013; Aslanidi et al., 2012). The wild type (WT) andall mutant rAAV3 vectors carrying the CBAp-driven enhanced greenfluorescence protein (EGFP) reporter gene (FIG. 40A) were used toevaluate their transduction efficiencies in a human HCC cell line, Huh7,under identical conditions. A summary of these data is provided in FIG.41. The transduction efficiency of two K-mutants (K528E; K533E) wasreduced >10-fold, and that of several Y- and T-mutants (Y272F; Y444F;T251V; Y705+731F+T492V) was reduced >2-fold. The transduction efficiencyof the rest of the mutants was increased, which ranged between <2-foldto >10-fold. The seven best mutants as well as the WT rAAV3 vectorscarrying the CBAp-driven FLuc reporter gene were then used to transduceHuh7 cells under identical conditions. These results (shown in FIG. 37A)indicated that the transduction efficiency of Y705+731F andS663V+T492V+K533R mutants was increased by ˜10-fold, and that ofS663V+T492V mutants was increased by ˜15-fold, compared with the WTrAAV3 vectors. To further validate the observed increased transductionefficiency of these mutants, the three best mutant rAAV3 vectorscarrying the CBAp promoter-driven EGFP reporter gene were used totransduce a different human HCC cell line, HepG2, under identicalconditions. The results (shown in FIG. 37B), demonstrated that thetransduction efficiency of S663V+T492V+K533R and Y705+731F mutants wasincreased by ˜2- and ˜3-fold, respectively, and that of S663V+T492Vmutants was increased by ˜5-fold, compared with the WT rAAV3 vectors.The transduction efficiency of the best mutant (S633V+T492V) was alsoevaluated in a more recently-established, human hepatoblastoma (HB) cellline, Hep293TT (Cheng et al., 2012) (FIG. 37C). Thus, the optimizedrAAV3 vector may prove useful in the potential gene therapy of humanliver cancer.

Modifications of Specific Amino Acids on rAAV3 Capsids do not Alter theVirus-Cellular Receptor Interaction.

Owing to the uncertainty of viral capsid amino acid(s) responsible forvirus-receptor(s) interaction, the inventors also examined whethercellular heparan sulfate proteoglycan (HSPG) and human hepatocyte growthfactor receptor (hHGFR), previously identified as receptor (Rabinowitzet al., 2004) and co-receptor (Ling et al., 2010) of WT rAAV3 vectors,were involved in transduction by our optimized rAAV3 vectors. Thefollowing three sets of studies were performed. First, transduction ofHuh7 cells with rAAV3-CBAp-EGFP vectors or the two best mutant vectorswere performed in the presence of either low (100 ng/mL) or high (100μg/mL) doses of soluble heparin. These results (shown in FIG. 37D,indicated that both Y705+731F and S663V+T492V mutants performed in asimilar manner as the WT rAAV3 vectors, in which the low-dose of heparinenhanced viral vector-mediated transduction efficiency, whereas thehigh-dose dramatically reduced it. Second, transduction assays wereperformed in the presence of 5 μg/mL human hepatocyte growth factor(hHGF), which was previously shown to significantly inhibit thetransduction efficiency of WT rAAV3 vectors (Ling et al., 2010). Theseresults, shown in FIG. 37E, demonstrated that the transductionefficiency of both the WT and the mutant viral vectors was significantlyaffected. And third, the WT and the two mutant rAAV3 vectors were usedto transduce a human breast cancer cell line, T47D, that expressesundetectable levels of endogenous hHGFR, as well as T47D cells stablytransfected with a hHGFR expression plasmids (T47D+hHGFR). Theseresults, shown in FIG. 37F, indicated that both the WT and the mutantrAAV3 vectors transduce T47D+hHGFR cells more efficiently (>5-fold) thanthe parental T47D cells. Taken together, these data confirmed that theoptimized rAAV3 vectors also utilize cellular HSPG and hHGFR asreceptor/co-receptor for their transduction.

Modifications of Specific Amino Acids on rAAV3 Capsids Further EnhanceViral Transduction Efficiency In Vivo Following Systemic Administration.

The inventors next evaluated the transduction efficiency of the twooptimized rAAV3 vectors in a murine xenograft model in vivo. To thisend, human Huh7 or Hep293TT tumor-bearing NSG mice were used (n=4), anda relatively low dose (5×10¹⁰ vgs/mouse) of rAAV3-Y705+731F-CBAp-FLuc orrAAV3-S663V+T492V-CBAp-FLuc vectors were delivered via the tail-vein.Whole-body bioluminescent imaging was performed 3-days' post-vectorinjections. From the results shown in FIG. 38A, it was clear that bothHuh7 and Hep293TT tumors were efficiently targeted by the optimizedrAAV3 vectors, and that transgene expression in both types of tumors wassignificantly enhanced by rAAV3-S663V+T492V vectors (FIG. 38B), comparedwith rAAV3-Y705+731F vectors, which have previously been shown to besignificantly more efficient than the WT rAAV3 vectors in vivo (Cheng etal., 2012). Three days' post-vector injections, mice were sacrificed,and liver and tumor tissues were harvested. Total DNA samples wereisolated from both liver and tumor tissues, and subjected to qPCRanalyses to determine the vector-genome copy numbers. From the datashown in FIG. 38C and FIG. 38D, it was apparent that the persistingvector genomes of rAAV3-S663V+T492V mutant in the tumor wassignificantly higher than those of rAAV3-Y705+731F mutant (FIG. 38C),presumably due to more efficient intracellular trafficking and nuclearentry (Aslanidi et al., 2013), which also correlates well with the FLuctransgene expression (FIG. 38A and FIG. 38B). No significant differencein the persisting vector genomes of the two vectors in the liver wasobserved, which is also comparable to that reported previously(Zincarelli et al., 2008). It is noteworthy, however, that despite thepresence of a roughly 5-fold higher vector genome copy numbers in theliver, compared with the tumor, little transgene expression in the liverwas detected with either of the two optimized rAAV3 vectors.

Suppression of tumorigenesis in the human liver cancer xenograft modelin vivo following systemic administration of optimized rAAV3 vectorsexpressing a novel therapeutic gene. All of the studies described thusfar were carried out with reporter genes, which lack therapeutic value.Thus, although the use of a number of well-established pro-apoptotic and“suicide” genes was contemplated, efforts were focused on anewly-identified therapeutic gene, which encodes trichosanthin (TCS), aribosome-inactivating protein, isolated from a traditional Chinesemedicinal herb, Trichosanthes kirilowii (Sha et al., 2013). Although thenucleotide sequence of the TCS gene was determined more than 20 yearsago (Shaw et al., 1994), the delivery of a gene encoding TCS into cellshas never been pursued. TCS gene-expressing cassettes under the controlof the AFPp were synthesized (detailed FIG. 40A), whose intracellularexpression significantly inhibited the growth of human HCC cell lines invitro.

(SEQ ID NO: 26) ATGATCAGATTCTTAGTCCTCTCTTTGCTAATTCTCACCCTCTTCCTAACAACTCCTGCTGTGGAGGGCGATGTTAGCTTCCGTTTATCAGGTGCAACAAGCAGTTCCTATGGAGTTTTCATTTCAAATCTGAGAAAAGCTCTTCCAAATGAAAGGAAACTGTACGATATCCCTCTGTTACGTTCCTCTCTTCCAGGTTCTCAACGCTACGCATTGATCCATCTCACAAATTACGCCGATGAAACCATTTCAGTGGCCATAGACGTAACGAACGTCTATATTATGGGATATCGCGCTGGCGATACATCCTATTTTTTCAACGAGGCTTCTGCAACAGAAGCTGCAAAATATGTATTCAAAGACGCTATGCGAAAAGTTACGCTTCCATATTCTGGCAATTACGAAAGGCTTCAAACTGCTGCAGGCAAAATAAGGGAAAATATTCCGCTTGGACTCCCTGCTTTGGACAGTGCCATTACCACTTTGTTTTACTACAACGCCAATTCTGCTGCGTCGGCACTTATGGTACTCATTCAGTCGACGTCTGAGGCTGCGAGGTATAAATTTATTGAGCAACAAATTGGGAAGCGTGTTGACAAAACCTTCCTACCAAGTTTAGCAATTATAAGTTTGGAAAATAGTTGGTCTGCTCTCTCCAAGCAAATTCAGATAGCGAGTACTAATAATGGACAGTTTGAAAGTCCTGTTGTGCTTATAAATGCTCAAAACCAACGAGTCACGATAACCAATGTTGATGCTGGAGTTGTAACCTCCAACATCGCGTTGCTGCTGAATAGAAACAATATGGCAGCCATGGATGACGATGTTCCTATGACACAGAGCTTTGGATGTGGAAGTTATGCTATTTAG (SEQ ID NO: 27)GAATTCATGATCAGATTCTTAGTCCTCTCTTTGCTAATTCTCACCCTCTTCCTAACAACTCCTGCTGTGGAGGGCGATGTTAGCTTCCGTTTATCAGGTGCAACAAGCAGTTCCTATGGAGTTTTCATTTCAAATCTGAGAAAAGCTCTTCCAAATGAAAGGAAACTGTACGATATCCCTCTGTTACGTTCCTCTCTTCCAGGTTCTCAACGCTACGCATTGATCCATCTCACAAATTACGCCGATGAAACCATTTCAGTGGCCATAGACGTAACGAACGTCTATATTATGGGATATCGCGCTGGCGATACATCCTATTTTTTCAACGAGGCTTCTGCAACAGAAGCTGCAAAATATGTATTCAAAGACGCTATGCGAAAAGTTACGCTTCCATATTCTGGCAATTACGAAAGGCTTCAAACTGCTGCAGGCAAAATAAGGGAAAATATTCCGCTTGGACTCCCTGCTTTGGACAGTGCCATTACCACTTTGTTTTACTACAACGCCAATTCTGCTGCGTCGGCACTTATGGTACTCATTCAGTCGACGTCTGAGGCTGCGAGGTATAAATTTATTGAGCAACAAATTGGGAAGCGTGTTGACAAAACCTTCCTACCAAGTTTAGCAATTATAAGTTTGGAAAATAGTTGGTCTGCTCTCTCCAAGCAAATTCAGATAGCGAGTACTAATAATGGACAGTTTGAAAGTCCTGTTGTGCTTATAAATGCTCAAAACCAACGAGTCACGATAACCAATGTTGATGCTGGAGTTGTAACCTCCAACATCGCGTTGCTGCTGAATAGAAACAATATGGCAGCCATGGATGACGATGTTCCTATGACACAGAGCTTTGGATGTGGAAGTTATGCTATTCTCGAGGACTACAAGGATGACGATGACAAGGATTACAAAGACGACGATGATAAGGACTATAAGGATGATGACGACAAATAA

Then, rAAV3-S663V+T492 mutant vectors were generated carrying this noveltherapeutic gene. In addition, to allow initiating the treatment at anearly time-point, before the tumors are palpable, a genetically-modifiedhuman HCC cell line, Huh7-FLuc, was also generated in which the FLucgene under the control of the CBAp promoter is stably transfected, whichalso allowed for monitoring the tumor growth by whole bodybioluminescent imaging. NSG mice (n=10) were subcutaneously injected onthe ventral side between shoulder blades with 5×10⁶ Huh7-FLuc cells.Four weeks post-xenografts, mice were divided into 2 groups, and 5×10¹⁰vgs of rAAV3-S663V+T492V-AFPp-TCS vectors were injected via thetail-vein in the first group (Day 0). The second group of mice wasinjected with 5×10¹⁰ vgs of rAAV3-S663V+T492V-AFPp-EGFP vectors to serveas appropriate controls. Whole-body bioluminescent imaging of mice wasperformed at Day 0, Day 3, Day 8, and Day 11 postvector-administrations. These results, shown in FIG. 39A, document thatwhereas Huh7-FLuc tumors grew progressively in mice injected withrAAV3-S663V+T492V-AFPp-EGFP vectors, tumor growth in mice injected withrAAV3-S663V+T492V-AFPp-TCS vectors was significantly inhibited up untilDay 11 (p<0.05), with maximal growth inhibition at Day 8 (p<0.01). Wholebody bioluminescent images of mice performed at Day 8 postvector-administrations are shown in FIG. 39B. Moreover, on Day 11, allmice were sacrificed, and serum levels of aspartate transaminase (AST)and alanine transaminase (ALT) were determined. No significantdifferences were observed between TCS-treated and control groups,suggesting little liver injury in mice (FIG. 39C).

Discussion

As stated above, HCC, which ranks fifth among solid tumors in humans,leads to ˜695,900 deaths worldwide each year. Although patients withearly diagnosis of HCC may benefit from surgical and/or chemotherapeuticinterventions, relapse of the disease is a frequent occurrence, and therate of treatment failure is high. For patients diagnosed with advancedHCC, the options are limited largely to supportive care. Although the USFood and Drug Administration (FDA) has approved the use of Sorafenib inpatients with advanced HCC, the median survival is increased by only ˜3months. Thus, it is readily clear that the development of noveltherapeutic options for HCC is sorely needed, and the combination ofgene therapy with tradition Chinese medicine (TCM) is a promisingoption. TCM medicine, as a main complementary and alternative medicinaltherapy, has already become a commonly-used treatment for HCC in China(Zhai et al., 2013). Recently, it has been reported that bioactivemonomeric compounds extracted from TCM herbs have the ability tosignificantly enhance the therapeutic efficiency mediated by rAAVvectors (Zhang et al., 2010; Mitchell et al., 2013; Wang et al., 2014;Ling et al., 2014). Here, a novel strategy has been developed thatcombines gene therapy with TCM administration, in which a therapeuticsuicide gene isolated from herbs is systemically delivered intomalignant cells in vivo through rAAV vectors.

Recombinant AAV vector-mediated gene therapy of HCC has been attemptedin the past. For example, the use of conventional, single-stranded (ss)rAAV2 vectors to target HCC in vivo has been reported (Su et al., 2000),but the transduction efficiency of these vectors was low. In subsequentstudies (Peng et al., 2000), no transduction was observed in tumorslarger than 2 mm following systemic administration. In more recentstudies, the use of rAAV8 vectors to mediate delivery of specificmiRNA-26A and miRNA-122 in mouse endogenous HCC tumor models was shownto result in inhibition of tumor growth (Kota et al., 2009; Hsu et al.,2012). However, rAAV8 vectors have a broad tropism to normal tissuesother than the liver in murine models (Zincarelli et al., 2008; Gao etal., 2002; Wang et al., 2005) and in non-human primates (Nathwani, etal., 2006; Nathwani et al., 2007). This example demonstrates that theremarkable tropism of rAAV3 vectors for human liver cancer cells invitro can also be exploited to achieve targeted delivery of thesevectors to human liver tumors in a xenograft mouse model in vivo. Inaddition, site-directed mutagenesis of specific amino acid residues onthe rAAV3 capsid can further augment the transduction efficiency ofrAAV3 vectors. Furthermore, the optimized rAAV3 vectors expressing anovel therapeutic gene can also be used to suppress human livertumorigenesis in a murine xenograft model. It should be emphasized thatthese studies were carried out with well-established tumors, and thedeliberate use of low vector doses to establish tumor-targeting. Thus,it is highly likely that the use of high vector doses and/or earlierintervention, before the tumor is well-established, it would be possibleto achieve a more desirable therapeutic endpoint. It is also tempting tospeculate that pending successful completion of additional studies withprimary human liver tumor xenografts, especially in livermicroenvironment, and safety and efficacy in large animal models,rAAV3-S663V+T492V vectors might prove useful in the potential genetherapy of human liver cancers.

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It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

All references, including publications, patent applications and patentscited herein are specifically incorporated herein by reference in theirentirety to the same extent as if each reference was individually andspecifically indicated to be incorporated by reference and was set forthin its entirety herein.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable order,unless otherwise indicated herein, or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illustrate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising,” “having,” “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of,”“consists essentially of,” or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents that are chemically and/or physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1. An rAAV3 vector comprising a modified capsid protein that comprises:(a) a non-tyrosine amino acid residue at one or more positionscorresponding to Y252, Y272, Y444, Y701, Y705, and Y731 of the wild-typeAAV3 capsid protein as set forth in SEQ ID NO:3; (b) a non-serine aminoacid residue at each of one or more positions corresponding to S459 orS663, of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3;(c) a non-threonine amino acid residue at each of one or more positionscorresponding to T251 or T492 of the wild-type AAV3 capsid protein asset forth in SEQ ID NO:3; (d) a non-lysine amino acid residue at each ofone or more positions corresponding to K528, K533, or K545 of thewild-type AAV3 capsid protein as set forth in SEQ ID NO:3; (e) (i) anon-tyrosine amino acid residue at position Y701, or Y705; and (ii) anon-tyrosine amino acid residue at position Y705 or Y731, or anon-serine amino acid residue at position S663 of the wild-type AAV3capsid protein as set forth in SEQ ID NO:3; (f) a combination of threeor more amino acid substitutions listed in (a), (b), (c), and (d); eachwith a non-native amino acid; (g) a combination of four or more aminoacid substitutions listed in (a), (b), (c), and (d); each with anon-native amino acid; or (h) a combination of five or more amino acidsubstitutions listed in (a), (b), (c), and (d); each with a non-nativeamino acid; or alternatively, wherein each of the amino acidsubstitutions is at an equivalent amino acid position correspondingthereto in any one of the other wild-type vector serotypes selected fromthe group consisting of AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, andAAV10, as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10,respectively.
 2. The rAAV3 vector in accordance with claim 1, whereinthe non-serine amino acid residue is selected from the group consistingof phenylalanine (F), valine (V), histidine (H), isoleucine (I), alanine(A), leucine (L) aspartic acid (D), asparagine (N). glutamic acid (E),arginine (R), and isoleucine (I); or the non-tyrosine, non-lysine, ornon-threonine amino acid residue is selected from the group consistingof serine (S), phenylalanine (F), valine (V), histidine (H), isoleucine(I), alanine (A), leucine (L) aspartic acid (D), asparagine (N).glutamic acid (E), arginine (R), and isoleucine (I).
 3. The rAAV3 vectorin accordance with claim 1, wherein the combination of three or moreamino acid substitutions include a non-native amino acid substitution atone or more of the following combination of acid residues: (a) Y701F,Y705F, and Y731F; (b) Y705F, Y731F, and S663V; (c) Y705F, Y731F, andT492V; (d) Y705F, Y731F, K533R; (e) S663V, T492V, and K533R; (f) Y705F,Y731F, S663V, and T492V; or (g) Y705F, Y731F, S663V, T492V, and K533R,of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3, or atthe equivalent surface-exposed amino acid residues in any one of thecorresponding wild-type AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,or AAV10 capsid proteins, as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10, respectively, or in any combination thereof. 4.The rAAV3 vector in accordance with claim 1, wherein the transductionefficiency of a virion comprising the vector is about 2- to about50-fold higher in a selected mammalian host cell than that of a virionthat comprises a corresponding, unmodified, rAAV3 vector.
 5. The rAAV3vector in accordance claim 1, wherein the transduction efficiency of avirion comprising the vector is about 6- to about 40-fold higher in aselected mammalian host cell than that of a virion that comprises acorresponding, unmodified, rAAV3 vector.
 6. The rAAV3 vector inaccordance with claim 1, wherein the transduction efficiency of a virioncomprising the vector is about 8- to about 30-fold higher in a selectedmammalian host cell than that of a virion that comprises acorresponding, unmodified, rAAV3 vector.
 7. The rAAV3 vector inaccordance with claim 1, wherein the virion comprising the vector isless susceptible to ubiquitination when introduced into a mammalian cellthan that of a virion that comprises a corresponding, unmodified, rAAV3vector.
 8. The rAAV3 vector in accordance with claim 1, wherein thevector further comprises a nucleic acid segment that encodes adiagnostic, therapeutic, or chemotherapeutic agent operably linked to apromoter capable of expressing the nucleic acid segment in a suitablehost cell comprising the vector.
 9. The rAAV3 vector in accordance withclaim 8, wherein the nucleic acid segment further comprises an enhancer,a post-transcriptional regulatory sequence, a polyadenylation signal, orany combination thereof, operably linked to the nucleic acid segment.10. The rAAV3 vector in accordance with claim 8, further comprising atleast a first mammalian intron sequence operably linked to the nucleicsegment.
 11. The rAAV3 vector in accordance with claim 8, wherein thepromoter is a heterologous promoter, a tissue-specific promoter, acell-specific promoter, a constitutive promoter, an inducible promoter,or any combination thereof.
 12. The rAAV3 vector in accordance withclaim 10, wherein the promoter is a liver-specific promoter, a tumorcell-specific promoter, or a combination thereof.
 13. The rAAV3 vectorin accordance with claim 8, wherein the nucleic acid segment expressesor encodes a polypeptide, a peptide, a ribozyme, a peptide nucleic acid,an siRNA, an RNAi, an antisense oligonucleotide, an antisensepolynucleotide, an antibody, an antigen binding fragment, or anycombination thereof.
 14. The rAAV3 vector in accordance with claim 8,wherein the at least a first nucleic acid segment encodes achemotherapeutic agent.
 15. The rAAV3 vector in accordance with claim 8,wherein the diagnostic, therapeutic or chemotherapeutic agent is anagonist, an antagonist, an anti-apoptosis factor, an inhibitor, areceptor, a cytokine, a cytotoxin, an erythropoietic agent, aglycoprotein, a growth factor, a growth factor receptor, a hormone, ahormone receptor, an interferon, an interleukin, an interleukinreceptor, a nerve growth factor, a neuroactive peptide, a neuroactivepeptide receptor, a protease, a protease inhibitor, a proteindecarboxylase, a protein kinase, a protein kinase inhibitor, an enzyme,a receptor binding protein, a transport protein or an inhibitor thereof,a serotonin receptor, or an uptake inhibitor thereof, a serpin, a serpinreceptor, a tumor suppressor, a cytotoxic agent, a cytostatic agent, ananti-inflammatory agent, or any combination thereof.
 16. The rAAV3vector in accordance with claim 1, comprised within an adeno-associatedviral particle or infectious rAAV3 virion. 17-29. (canceled)
 30. Amethod for providing a mammal in need thereof with a diagnostically- ortherapeutically-effective amount of a selected biological molecule, themethod comprising providing to a cell, tissue or organ of a mammal inneed thereof, an amount of the rAAV3 vector in accordance with claim 1;and for a time effective to provide the mammal with a diagnostically- ora therapeutically-effective amount of the selected biological molecule.31. A method for diagnosing, preventing, treating, or ameliorating atleast one or more symptoms of liver cancer, including HCC, in a mammal,the method comprising, administering to a mammal in need thereof therAAV3 vector in accordance with claim 1, in an amount and for a timesufficient to diagnose, prevent, treat or ameliorate the one or moresymptoms of the liver cancer in the mammal.
 32. The method in accordancewith claim 31, wherein the mammal is human.
 33. A method of transducinga population of liver cells or liver tumor cells in a human diagnosedwith, having, or suspected of having HCC; the method comprisingadministering to the human, a composition that comprises an effectiveamount of the rAAV3 vector in accordance with claim 1, for a timeeffective to transduce the population of liver cells or liver tumorcells.